U.S. patent number 11,085,038 [Application Number 16/843,299] was granted by the patent office on 2021-08-10 for polypeptide library.
This patent grant is currently assigned to MORPHOSYS AG. The grantee listed for this patent is MorphoSys AG. Invention is credited to Andreas Bultmann, Markus Moosmeier, Roger Muller, Josef Prassler.
United States Patent |
11,085,038 |
Muller , et al. |
August 10, 2021 |
Polypeptide library
Abstract
The invention relates to novel polypeptide libraries that are
conformationally constrained in an anti-parallel, helix-turn-helix
arrangement. The invention further relates to methods of generating
and screening such libraries for biological, pharmaceutical and
other uses.
Inventors: |
Muller; Roger (Munich,
DE), Bultmann; Andreas (Planegg, DE),
Prassler; Josef (Germering, DE), Moosmeier;
Markus (Landau a.d. Isar, DE) |
Applicant: |
Name |
City |
State |
Country |
Type |
MorphoSys AG |
Planegg |
N/A |
DE |
|
|
Assignee: |
MORPHOSYS AG (Planegg,
DE)
|
Family
ID: |
55484880 |
Appl.
No.: |
16/843,299 |
Filed: |
April 8, 2020 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20200231963 A1 |
Jul 23, 2020 |
|
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
16080738 |
|
10655125 |
|
|
|
PCT/EP2017/055002 |
Mar 3, 2017 |
|
|
|
|
Foreign Application Priority Data
|
|
|
|
|
Mar 4, 2016 [EP] |
|
|
16158782 |
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C40B
40/02 (20130101); C12N 15/1037 (20130101); C40B
40/10 (20130101) |
Current International
Class: |
C40B
40/10 (20060101); C12N 15/10 (20060101); C40B
40/02 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
WO94/29332 |
|
Dec 1994 |
|
WO |
|
WO00/27878 |
|
May 2000 |
|
WO |
|
Other References
Bryson et al (Protein Science 7:1404-14) (Year: 1998). cited by
examiner .
Fujii, I. "Beyond antibodies: generation of conformationally
constrained peptides for molecular-targeting therapy" Yakugaku
Zasshi 2009 129:1303-1309. cited by applicant .
Fujii et al. "A conformationally purified .alpha.-helical peptide
library" Tetrahedron Letters 2001 42:3323-3325. cited by applicant
.
Fujii et al. "Beyond antibodies: directed evolution of
molecular-targeting peptides in phage-displayed libraries of
conformationally constrained peptides" Drug Delivery System 2011
26:593-603. cited by applicant .
Fujiwara & Fujii "Phage selection of peptide "microantibodies""
Current Protocols in Chemical Biology 2013 5:171-194. cited by
applicant .
Fujiwara et al. "Selection of inhibitory peptides for Aurora-A
kinase from a phage-displayed library of helix-loop-helix peptides"
Bioorganic and Medicinal Chemistry Letters 2010 20:1776-1778. cited
by applicant .
Harrison et al. "Downsizing human, bacterial, and viral proteins to
short water-stable alpha helices that maintain biological potency"
Proc. Natl. Acad. Sci. USA. 2010 107:11686-11691. cited by
applicant .
Landschultz et al. "The leucine zipper: a hypothetical structure
common to a new class of DNA binding proteins" Science 1988
240:1759-1764. cited by applicant .
Matsubara et al. "Selection of a carbohydrate-binding domain with a
helix-loop-helix structure" Biochemistry 2008 47:6745-6751. cited
by applicant .
McFarlane et al. "The use of coiled-coil proteins in drug delivery
systems" European Journal of Pharmacology 2009 625:101-107. cited
by applicant .
O'Shea et al. "Evidence that the leucine zipper is a coiled coil"
Science 1989 242:538-542. cited by applicant .
O'Shea et al. "Preferential heterodimer formation by isolated
leucine zippers from fos and jun" Science 1989 245:646-648. cited
by applicant .
Parry et al. "Fifty years of coiled-coils and .alpha.-helical
bundles: A Close relationship between sequence and structure"
Journal of Structural Biology 2008 163:258-269. cited by applicant
.
Rudert et al. "A phage-based system to select multiple
protein-protein interactions simultaneously from combinatorial
libraries" FEBS Letters 1998 440:135-140. cited by applicant .
International Search Report and Written Opinion in
PCT/EP2017/055002 dated May 17, 2017. cited by applicant .
Cohen, C. and Parry, A.D. ".alpha.-Helical Coiled Coils and
Bundles: How to Design an .alpha.-Helical Protein" Proteins:
Structure, Function and Genetics 1990 7:1-15. cited by applicant
.
Eisenberg et al. "Analysis of Membrane and Surface Protein
Sequences with the Hydrophobic Moment Plot" J. Mol. Biol. 1984
179:125-142. cited by applicant .
Harbury et al. "High Resolution Protein Design with Backbone
Freedom" Science 1998 282:1462-1467. cited by applicant .
Kohn, W.D. and Hodges, R.S. "De novo design of .alpha.-helical
coiled coils and bundles: models for the development of
protein-design principles" TIBTECH 1998 16:379-389. cited by
applicant .
Lupas A. and Gruber, M. "The Structure of .alpha.-Helical Coiled
Coils" Advances in Protein Chemistry 2005 70:37-78. cited by
applicant .
Mason, J.M. and Arndt, K.M. "Coiled Coil Domains: Stability,
Specificity, and Biological Implications" ChemBioChem 2004
5:170-176. cited by applicant .
Pace, C.N. and Scholtz. J.M. "A Helix Propensity Scale Based on
Experimental Studies of Peptides and Proteins" Biophysical Journal
1998 76:422-427. cited by applicant .
Schneider et al. "Analysis and design of three-stranded coiled
coils and three-helix bundles" Folding & Design 1998 3:R29-R40.
cited by applicant .
Woolfson, D.N. "The Design of Coiled-Coil Structures and
Assemblies" Advances in Protein Chemistry 2005 70:79-112. cited by
applicant .
International Preliminary Examination Report on Patentability in
PCT/EP2017/055002 dated Sep. 4, 2018. cited by applicant .
Written Opinion in Singapore Application No. 11201806022R dated
Aug. 5, 2019. cited by applicant .
Pack et al. "Improved Bivalent Miniantibodies, with Identical
Avidity as Whole Antibodies, Produced by High Cell Density
Fermentation of Escherichia coli" Biotechnology 1993 11:1271-1277.
cited by applicant .
Office Communication dated Oct. 4, 2019 in U.S. Appl. No.
16/080,738, filed Aug. 29, 2018 cited by applicant .
Office Communication dated Jan. 13, 2020 in U.S. Appl. No.
16/080,738, filed Aug. 29, 2018 cited by applicant.
|
Primary Examiner: Gross; Christopher M
Attorney, Agent or Firm: Licata & Tyrrell P.C.
Claims
The invention claimed is:
1. A pharmaceutical composition comprising a polypeptide isolated
from a library of polypeptides, wherein each member of the library
comprises a helix-turn-helix scaffold structure of the formula
Helix-1-Li-Helix-2, wherein Helix-1 and Helix-2 comprise a first
and second .alpha.-helical peptide, wherein each of said
.alpha.-helical peptides comprises the amino acid sequence
TABLE-US-00008 (SEQ ID NO: 1)
X1-X2-Hy-Var1-X3-Hy-Var1-Var2-X4-Hy-Var1-X5-Hy- Var1-Var3,
wherein X1 is D, T, N or S, X2 is E, P, Q, W or D, X3 is M, A, I, Q
or R, X4 is A, L, R, M, K or E, X5 is M, L, A, W, F or K, Hy is any
amino-acid residue having a side chain exhibiting a hydrophobicity
of greater than 0.62, and Var1, Var2 and Var3 are each diversified
amino acid residues selected from mixtures of the natural occurring
amino acids, excluding G, P and C, Li is a linker, and said first
and said second .alpha.-helical peptide form an anti-parallel,
coiled-coil structure.
2. The pharmaceutical composition of claim 1 wherein the linker Li
comprises 1 to 30 amino acid residues (SEQ ID NO: 2).
3. The pharmaceutical composition of claim 1, wherein X1 is D, X2
is E, X3 is Q in Helix-1 and A in Helix-2, X4 is E in Helix-1 and K
in Helix-2, and X5 is K in Helix-1 and M in Helix-2 as depicted in
SEQ ID NO:3.
4. The pharmaceutical composition of claim 1, wherein Hy is L, V or
I as depicted in SEQ ID NO:4.
5. The pharmaceutical composition of claim 1, wherein Var2 is a
mixture of R, Q and E, and Var3 is a mixture of R, Q and H as
depicted in SEQ ID NO:5.
6. The pharmaceutical composition of claim 1, wherein polypeptide
is linked to at least one additional moiety.
7. The pharmaceutical composition of claim 6, wherein said
additional moiety is an antibody or antibody fragment thereof, a
toxin, a cytokine, a reporter enzyme, a moiety being capable of
binding a metal ion, a tag suitable for detection and/or
purification, a homo- or hetero-association domain, a moiety which
increases solubility of a protein, or a moiety which comprises an
enzymatic cleavage site.
8. The pharmaceutical composition of claim 1 further comprising at
least one pharmaceutically acceptable carrier.
9. A pharmaceutical composition comprising a helix-turn-helix
scaffold structure of formula Helix-1-Li-Helix-2, linked to an
antibody or an antibody fragment, wherein said helix-turn-helix
scaffold structure comprises a first and a second .alpha.-helical
peptide that form an anti-parallel, coiled-coil structure, wherein
each of said .alpha.-helical peptides comprises the amino acid
sequence TABLE-US-00009 (SEQ ID NO: 1)
X1-X2-Hy-Var1-X3-Hy-Var1-Var2-X4-Hy-Var1-X5-Hy- Var1-Var3,
wherein X1 is D, T, N, S or P, X2 is E, P, Q, W or D, X3 is M, A,
I, Q or R, X4 is A, L, R, M, K or E, X5 is M, L, A, W, F or K, Hy
is any amino-acid residue having a side chain exhibiting a
hydrophobicity of greater than 0.62, Var1, Var2 and Var3 are
mixtures of the natural occurring amino acids, excluding G, P and
C, and Li is a linker.
10. The pharmaceutical composition of claim 9 further comprising at
least one pharmaceutically acceptable carrier.
11. A method for preventing or treating a disorder or condition
associated with the undesired presence of a target molecule of
interest specifically bound by the polypeptide of the
pharmaceutical composition of claim 1 in a subject, said method
comprising administering to the subject the pharmaceutical
composition of claim 1.
12. The method of claim 11 wherein the disorder or condition
associated with the undesired presence of a target molecule of
interest is an autoimmune disease, inflammatory disease, cancer,
neovascular disease, infectious disease, thrombosis, myocardial
infarction or diabetes.
13. A method for preventing or treating a disorder or condition
associated with the undesired presence of a target molecule of
interest specifically bound by the polypeptide of the
pharmaceutical composition of claim 9 in a subject, said method
comprising administering to the subject a pharmaceutical
composition comprising a helix-turn-helix scaffold structure of
formula Helix-1-Li-Helix-2, linked to an antibody or an antibody
fragment, wherein said helix-turn-helix scaffold structure
comprises a first and a second .alpha.-helical peptide that form an
anti-parallel, coiled-coil structure, wherein each of said
.alpha.-helical peptides comprises the amino acid sequence
TABLE-US-00010 (SEQ ID NO: 1)
X1-X2-Hy-Var1-X3-Hy-Var1-Var2-X4-Hy-Var1-X5-Hy- Var1-Var3,
wherein X1 is D, T, N, S or P, X2 is E, P, Q, W or D, X3 is M, A,
I, Q or R, X4 is A, L, R, M, K or E, X5 is M, L, A, W, F or K, Hy
is any amino-acid residue having a side chain exhibiting a
hydrophobicity of greater than 0.62, Var1, Var2 and Var3 are each
diversified amino acid residues selected from mixtures of the
natural occurring amino acids, excluding G, P and C, and Li is a
linker; and wherein the disorder or condition associated with the
undesired presence of a target molecule of interest is an
autoimmune disease, inflammatory disease, cancer, neovascular
disease, infectious disease, thrombosis, myocardial infarction or
diabetes.
Description
This patent application claims the benefit of priority from U.S.
application Ser. No. 16/080,738, filed Aug. 29, 2018, which is the
U.S. National Stage of International Application No.
PCT/EP2017/055002, filed Mar. 3, 2017, which claims the benefit of
priority from EP 16158782.9, filed Mar. 4, 2016, teachings of each
of which are incorporated by reference in their entirety.
FIELD OF THE INVENTION
The invention relates to novel polypeptide libraries that are
conformationally constrained in an anti-parallel, helix-turn-helix
arrangement. The invention further relates to methods of generating
and screening such libraries for biological, pharmaceutical and
other uses.
BACKGROUND OF THE INVENTION
Peptide libraries have emerged as a powerful resource to identify
therapeutically relevant molecules. In addition, peptide libraries
are also relevant resources for many other purposes and for basic
research.
Therapeutically, peptides have certain advantages over small
molecules and large biologics, such as antibodies. As compared to
small molecules, peptides typically have a larger interaction
interface with an antigen, which comprises hydrogen bonds and van
der Waals forces. This leads to high affinity binding, a high
specificity for the antigen and typically a high potency. As
compared to antibodies, peptides are much smaller and therefore
typically penetrate tissue more easily. Certain tumors are
inaccessible for antibody therapy.
Numerous phage display peptide libraries do exist, including
libraries that utilize constrained peptides. Constraint peptides
overcome certain disadvantages that are associated with linear
peptides, including weak binding affinities due to a higher
conformational flexibility, and an increased susceptibility to
proteolytic degradation in the human body.
A natural occurring constrained motif of (poly)peptides is the
.alpha.-helical bundle. .alpha.-helices constitute the largest
class of protein secondary structures and play a major role in
mediating protein-protein interactions. However, short synthetic
peptides of 10-30 amino acids in length are usually not
thermodynamically stable helices in water and adopt only random
structures (Harrison et al., Proc. Natl. Acad. Sci. USA. 2010 Jun.
29; 107(26)).
.alpha.-helical bundles can appear in different forms, including
two, four, or even multiple bundles. The individual .alpha.-helical
peptides in such bundle proteins may be orientated in a parallel or
anti-parallel arrangement, thus forming coiled-coil structures in
which the helical axes are aligned slightly offset from one
another.
The .alpha.-helical structures that occur in such bundles usually
comprise heptad repeats with a profile consisting of a hydrophilic
exterior, a hydrophobic interior and a border of polar amino acid
residues that form interhelical salt bridging interactions.
Many natural occurring proteins, like keratin, myosin, epidermin,
fibrinogen and tropomysin, have a coiled-coil structure formed by
two dimerized .alpha.-helical peptides. Furthermore, coiled-coil
structures are frequently found on DNA binding proteins, where this
motif is referred to as a leucine zipper.
Coiled-coil domains are also found in the Jun, Fos (O'Shea et al.,
Science. 245:646-648 (1989)), C/EBP (Landschultz et al., Science.
240: 1759-1764 (1988)) and for instance in GCN4 binding proteins
(O'Shea et al., Science. 242:538-542 (1989)). Naturally occurring
.alpha.-helical coiled-coil structure are often found in a parallel
orientation, which is thought to be a stable conformation.
Approaches have been described to adapt such structures to design
specific recognition molecules. WO94/29332 describes polypeptides
containing anti-parallel coiled-coils wherein said scaffolds were
modified to incorporate helical recognition sequences from
naturally-occurring proteins such as DNA binding proteins and
cytokines.
U.S. Pat. No. 5,824,483 describes the construction of a de novo
designed and chemically synthesized combinatorial library of
.alpha.-helical peptides. The .alpha.-helical peptides were
stabilized by intrahelical lactam bridges and optionally by an
additional second .alpha.-helical peptide thus resulting in a
dimeric coiled-coil structure in a parallel or anti-parallel
orientation. However, the only enabled peptide library encompassed
a single 24 amino acid long .alpha.-helical polypeptide chain which
is stabilized via two intrahelical lactam bridges and which is
diversified at 5 amino acid positions.
Fujii et al. (Tetrahedron Letters 42, 3323-3325 (2001)) describes a
more specific approach for a helix-turn-helix based library. The
scientific publication discloses a de novo chemically synthesized
anti-parallel orientated helix-turn-helix peptide library wherein
the amino- and carboxyl-terminal peptides are linked via a glycine
based linker. Each of the two helix-turn-helix forming peptides
consisted of 14 amino acids and was stabilized by hydrophobic
interactions with leucine residues on the two respective helices.
In contrast to the library of the present disclosure, only the
carboxyl-terminal helix peptide was diversified at 3 solvent
exposed positions with a mixture of 5 naturally occurring amino
acid residues.
A complementary method for utilizing a peptide library is the
display of such libraries on filamentous bacteriophages. This
method allows the preparation of libraries as large as 10.sup.10
unique peptide members, many orders of magnitude larger than
libraries that may be prepared synthetically.
A phage displayed anti-parallel orientated helix-turn-helix peptide
library was described by Fujii and Coworkers in 2008 (Biochemistry,
47, 6745-6751 (2008)). In contrast to the above mentioned library,
the carboxyl-terminal helix peptide was randomized at 5 solvent
exposed regions yielding in a theoretical library size of
3.2.times.10.sup.6. The library was displayed on the major coat
protein VIII of filamentous phage with a glycine/serine linker in
conjunction with a detectable tag. A particular utilization of the
helix-turn-helix peptide library to generate "Microantibodies" has
been further described by Fujii et al. in 2011 (Drug Delivery
System, 26-6, 2011, p. 593-603), in 2009 (Yakugaku Zasshi, 129
(11), 1303-1309, 2009) and 2013 (Current Protocols in Chemical
Biology, vol. 5 (3), 171-194, 2013)
A common structural feature of the two libraries described by Fujii
and Coworkers is the predominant usage of alanine at solvent
exposed positions of the two .alpha.-helical peptides. Stretches of
alanine (poly alanine) are known to facilitate formation of
.alpha.-helical structures but they also may display low solubility
in aqueous solutions and thus are prone for aggregation.
More importantly, the libraries described by Fujii and Coworkers
are only diversified within the carboxyl-terminal .alpha.-helical
peptide by diversifying solvent exposed alanine positions. In this
scenario, the non-diversified amino-terminal peptide is thought to
retain its .alpha.-helical structure and to stabilize the
helix-turn-helix structure of the molecule. However, randomization
of the carboxyl-terminal .alpha.-helical peptide as provided by
Fujii still resulted in library members with undesired multiple
random like conformations which required a particular purification
process in order to enrich for correctly folded helix-turn-helix
structures (Fujii et al. (Tetrahedron Letters 42, 3323-3325
(2001)).
A major disadvantage of diversifying only one of the two
.alpha.-helical peptides lies in the fact that the approach
significantly limits the achievable library size and significantly
narrows down the interaction interphase between the polypeptides of
the library and their bound target molecules of interest resulting
in reduced specific and affinity.
Based on limitations of the above mentioned approaches, there is
still an unmet need to develop improved helix-turn-helix
polypeptide libraries of considerable size.
The library of the present disclosure differs in multiple ways from
the libraries disclosed by Fujii. The design of the library of the
present disclosure is based on a combined approach taking into
account statistical, structural and rational factors. This included
in a first instance the analysis and use of the most abundant amino
acid residues found at given positions in natural occurring
.alpha.-helical structures. Such amino acids are considered to have
favorable biophysical properties including low immunogenicity,
resistance against temperature and chemical denaturation, relative
insensitiveness for pH alterations, serum stability and resistance
against proteolytic degradation by proteases.
Secondly, the variable positions within the helix-turn-helix
library of the present disclosure are present on both, the amino-
and the carboxyl terminal .alpha.-helical peptide and are displayed
in the same relative parallel orientation. This two features enable
the formation of a wide and flat interaction interface over the
whole length of the helix-turn-helix molecule. The enlarged
interaction interface is crucial for an optimal protein-protein
interaction with a target antigen of interest resulting in improved
specificity and affinity, both critical aspect in the development
of therapeutic molecules.
Furthermore, in order to prevent a potential destabilization of the
helix-turn-helix scaffold caused by the introduction of a large
number of variable positions in both .alpha.-helical peptides,
additional structural consideration for promoting helix formation
and stabilizing the helix-turn-helix structure were taken into
account to select the most appropriate amino acid residue at each
invariant position. These amino acid residues were selected to
stabilize the molecular structure by inter- and intrahelical
electrostatical interactions and interhelical hydrogen bonding.
In summary, the library of the present disclosure overcomes the
limitations of the helix-turn-helix libraries disclosed by Fujii
and Coworkers by maximizing the number of diversified positions
without compromising the stabilizing .alpha.-helical structures
leading to a more efficient development of the resulting
polypeptides and an increase in safety and efficacy of the
respective therapeutics in patients.
SUMMARY OF THE INVENTION
The present disclosure discloses a helix-turn-helix (HTH)
polypeptide library, which is characterized by an extraordinary
large library size. Polypeptides can be isolated from this library,
which bind to target molecules of interest with high affinity,
specificity, and functionality.
Preferably, said polypeptide library is a phage display
library.
The sequence variations of the library of the present disclosure is
present on both .alpha.-helical domains of the HTH scaffold. The
polypeptides encoded by the library may therefore bind to, for
example, a receptor with two or more spatially distinct but related
binding sites. The variable positions present on both
.alpha.-helical domains may further contribute to an enlarged
interaction interphase between a particular polypeptide and its
target antigen, thus resulting in improved specificity and
affinity.
In another aspect of the present disclosure, the diversified amino
acid residue positions are located on the solvent exposed regions
of the HTH scaffold as described herein.
The library of the present disclosure can be diversified in up to
12 amino acid positions, each with a mixture of up to 17 natural
occurring amino acid residues. Therefore, a library size of greater
than 1.times.10.sup.11 can be achieved.
It was also found that the polypeptides isolated from the library
have several superior properties over traditional immunoglobulin
and non-immunoglobulin binding agents. Such properties include for
instance their compact and small size (.about.6 kDa), low
immunogenicity, extreme stability against thermal and chemical
denaturation, relatively insensitiveness to changes in pH and to
proteolytic degradation.
The library of the present disclosure can be used to identify
molecules for therapeutic use, or can be used to characterize such
molecules by means, such as, epitope mapping.
In one aspect the present disclosure provides a library of
polypeptides, wherein each member of the library comprises a
helix-turn-helix scaffold structure of the formula
Helix-1-Li-Helix-2,
wherein Helix-1 and Helix-2 comprise a first and second
.alpha.-helical peptide, wherein each of said .alpha.-helical
peptides comprises the amino acid sequence
TABLE-US-00001 (SEQ ID NO: 1)
X1-X2-Hy-Var1-X3-Hy-Var1-Var2-X4-Hy-Var1-X5-Hy- Var1-Var3,
wherein X1 is D, T, N, S or P, X2 is E, P, Q, W or D, X3 is M, A,
I, Q or R, X4 is A, L, R, M, K or E, X5 is M, L, A, W, F or K, Hy
is any amino-acid residue having a side chain exhibiting a
hydrophobicity of greater than 0.62, and Var1, Var2 and Var3 are
mixtures of the natural occurring amino acids, excluding G, P and
C, Li is a linker, and said first and second .alpha.-helical
peptide form an anti-parallel, coiled-coil structure.
In yet a further aspect of the present disclosure the linker Li
comprises 1 to 30 amino acid residues (SEQ ID NO: 2).
In another embodiment of the present disclosure (SEQ ID NO: 3), X1
is D, X2 is E, X3 is Q in Helix-1 and A in Helix-2, X4 is E in
Helix-1 and K in Helix-2, and X5 is K in Helix-1 and M in
Helix-2.
In an embodiment, the present disclosure provides a library,
wherein Hy is L, V or I (SEQ ID NO: 4).
In an embodiment, the present disclosure provides a library (SEQ ID
NO: 5), wherein Var2 is a mixture of E, R and Q, and Var3 is a
mixture of R, Q and H.
In a further embodiment of the present disclosure, the polypeptides
of the library are displayed on bacteriophage.
In a further embodiment of the present disclosure, the library
comprises at least 1.times.10.sup.6 polypeptide members.
In a further embodiment of the present disclosure, each member of
the library is linked to at least one additional moiety.
In an embodiment of the present disclosure, said additional moiety
is an antibody or antibody fragment thereof, a toxin, a cytokine, a
reporter enzyme, a moiety being capable of binding a metal ion, a
tag suitable for detection and/or purification, a homo- or
hetero-association domain, a moiety which increases solubility of a
protein, or a moiety which comprises an enzymatic cleavage
site.
In a further embodiment, the present disclosure provides a
collection of nucleic acid molecules encoding the library members
of the present disclosure.
In a further embodiment, the present disclosure provides a vector
comprising the collection of nucleic acid molecules encoding the
library members of the present disclosure. In certain embodiments,
said vector is a display vector or an expression vector.
In a further embodiment, the present disclosure provides a host
cell comprising the collection of nucleic acid molecules or the
vector encoding the library members of the present disclosure.
In a further embodiment, the present disclosure provides a method
to isolate a polypeptide specific for an antigen, said method
comprising the steps of: a. contacting the library according to the
present disclosure with an antigen; b. removing those members of
the library which do not bind to the antigen; and c. recovering
those members of the library which did bind to the antigen.
In a further embodiment, the present disclosure provides a
polypeptide identified by the method(s) of the present
disclosure.
In an embodiment, the present disclosure provides a
helix-turn-helix scaffold structure linked to an antibody or an
antibody fragment, wherein said helix-turn-helix scaffold structure
comprises a first and a second .alpha.-helical peptide that form an
anti-parallel, coiled-coil structure.
DESCRIPTION OF THE DRAWINGS
FIG. 1 shows an illustration of the helix-turn-helix HTH-lib1
library of the present disclosure. FIG. 1 discloses SEQ ID NO: 1,
7, and 1, respectively, in order of appearance.
FIG. 2A shows a helical wheel cross section for the two
.alpha.-helical peptides of the helix-turn-helix reference sequence
(HTHdes2) of the present disclosure. The diversified amino acid
positions Var1, Var2, and Var3 within the HTH-lib1 library of the
present disclosure are denoted within the outer circles.
FIG. 2B depicts a three dimensional cartoon of the helix-turn-helix
HTH-lib1 library of the present disclosure, indicating the
positions of the variable amino-acid residues Var1, Var2 and
Var3.
FIG. 2C depicts the HTHdes2 reference sequence with intra- and
interchain electrostatic interactions as well as interchain
hydrogen bonding between distinct amino acid residues.
FIG. 3 shows the design of the polypeptide library HTH-lib1 as
disclosed herein (SEQ ID NO: 8 and 9, respectively, in order of
appearance).
FIGS. 4A and 4B shows a quality assessment of the polypeptide
library of FIG. 3. FIG. 4A shows the amino acid distribution at
diversified positions of the HTH-lib1 library of individually
sampled clones using Sanger sequencing. FIG. 4B depicts for each
diversified amino acid position the expected amino acid
distribution in context of the sequencing results of FIG. 4A.
FIG. 5 shows examples of polypeptides from the unselected HTH-lib1
library (SEQ ID NO: 8, 9, and 10 through 41, respectively, in order
of appearance). The examples illustrate that the design of the
library was successfully produced.
FIG. 6 shows a simplified view of the display and expression
vectors as well as the PCR based approach for subcloning a
polypeptide-encoding insert from the display vector into the
expression vector. FIG. 6 discloses "Hiss" as SEQ ID NO: 42.
FIG. 7 shows exemplary sequencing results derived from polypeptides
identified after an ELISA screening of individual clones derived
from a 2.sup.nd round panning output of the HTH-lib1 library of
FIG. 3 on Target-X. The result confirms that a diverse number of
target specific polypeptides can be identified (SEQ ID NO: 43
through 67, respectively, in order of appearance).
FIG. 8 shows two circular dichroism (CD) spectra of a Target-X
specific polypeptide in 10 mM phosphate buffer at pH 7.2 at
20.degree. C. and a polypeptide concentration of 0.1 mg/ml treated
at temperatures of 20.degree. C. and 90.degree. C.
FIG. 9, views A, B, C, D and E, show illustrations of HTH scaffolds
as disclosed herein, linked to antibodies or antibody fragments.
View A of FIG. 9 depicts two HTH scaffolds linked to the
carboxyl-terminus of the heavy chains of an antibody. View B of
FIG. 9 depicts two HTH scaffolds linked to the carboxyl-terminus of
the light chains of an antibody. View C of FIG. 9 depicts a HTH
scaffold linked to the carboxyl-terminus of the heavy chain of an
antibody Fab-fragment. View D and view E of FIG. 9 depicts an HTH
scaffold linked to the carboxyl- or amino-terminus of an
Fc-fragment of an antibody.
DEFINITIONS
The terms "comprising", "comprises" and "comprised of" as used
herein are synonymous with "including", "includes" or "containing",
"contains", and are inclusive or open-ended and do not exclude
additional, non-recited members, elements or method steps.
"Library" means an entity comprising more than one member. In the
context of the present disclosure, this term refers to a library of
polypeptides, wherein said library comprises at least two different
polypeptides.
By the term "peptide" is meant a short molecule having less than or
equal to 20 amino acids.
The term "polypeptide" means a molecule having more than 20 amino
acids.
A "fusion protein" is a polypeptide having two portions covalently
linked together, where each of the portions is a peptide or
polypeptide having a different property. The property may be a
biological property, such as activity in vitro or in vivo. The
property may also be a simple chemical or physical property, such
as binding to a target molecule, catalysis of a reaction, etc. The
two portions may be linked directly by a single peptide bond or
through a peptide spacer containing one or more ammo acid residues.
Generally, the two portions and the spacer will be in reading frame
with each other.
As used herein, the term "helix-turn-helix scaffold" or "HTH
scaffold" refers to a secondary structure of a polypeptide in which
two .alpha.-helices are orientated in a parallel or an
anti-parallel orientation, and in which the two .alpha.-helices are
linked via a short stretch of amino acids.
The terms "heptad", "heptad unit", "heptad repeat unit" and "heptad
motif" are used interchangeably herein and refer to a 7-mer peptide
fragment that is repeated two or more times within a HTH scaffold.
The tertiary structure of a .alpha.-helix is such that 7 amino acid
residues in the primary sequence correspond to approximately 2
turns of the .alpha.-helix. Accordingly, a primary amino acid
sequence giving rise to a .alpha.-helical conformation may be
broken down into units of 7 residues each. The individual positions
of a heptad unit are denoted by small letters, i.e. one heptad unit
is for example represented by the sequence `abcdefg`, `bcdefga`,
`cdefgab`, `defgabc`, `efgabcd`, `fgabcde` or `gabcdef`. The `a`
and the `d` position of a heptad unit assembled in an HTH scaffold
of the present disclosure are of hydrophobic nature. These
positions are typically either leucine, isoleucine or valine, and
the parallel or the anti-parallel secondary structure of the HTH
are formed by hydrophobic interactions via these positions between
different heptad units present on two distinct .alpha.-helical
peptides.
The terms "coiled-coil" and "coiled-coil structure" are used
interchangeably herein and will be clear to the person skilled in
the art based on the common general knowledge and the description
and further references cited herein. In general, a coiled-coil
structure is used by nature to stabilize .alpha.-helices in
proteins. A coiled-coil is a structural motif in polypeptides or
proteins in which 2 to 7 .alpha.-helices are coiled together. The
coiled-coil formation of .alpha.-helical peptides is facilitated
through a burial of hydrophobic side chains by arranging them on
one side of the .alpha.-helices so that they are not accessed by
polar water molecules. A typical coiled-coil motif (4-3 hydrophobic
repeat) is a heptad repeat of amino acids from `a` to `g` so that
`a` and `d` are hydrophobic. Particular reference in this regard is
made to review papers concerning coiled-coil structures, such as
for example, Cohen and Parry Proteins 1990, 7:1-15; Kohn and Hodges
Trends Biotechnol 1998, 16:379-389; Schneider et al Fold Des 1998,
3:R29-R40; Harbury et al. Science 1998, 282:1462-1467; Mason and
Arndt Chem Bio Chem 2004, 5:170-176; Lupas and Gruber Adv Protein
Chem 2005, 70:37-78; Woolfson Adv Protein Chem 2005, 70:79-112;
Parry et al. J Struct Biol 2008, 163:258-269; McFarlane et al. Eur
J Pharmacol 2009, 625:101-107.
As used herein, the term "anti-parallel" refers to an HTH scaffold
in which two .alpha.-helical peptides of an HTH scaffold are
arranged such that the amino-terminal end of one .alpha.-helical
peptide is aligned with the carboxyl-terminal end of the second
.alpha.-helical peptide, and vice versa. Thus, the relative
orientation of the heptad `a-g` positions of two interacting
.alpha.-helices aligned in an anti-parallel orientation is in the
opposite direction. For instance, if the heptad positions of a
first helix is defined as `abcdefg` as read from the amino- to the
carboxyl-terminus, the heptad positions of a second .alpha.-helix
in an anti-parallel orientation would be defined as `gfedcba` as
read from the amino- to the carboxyl-terminus.
As used herein, the term "parallel" refers to an HTH scaffold in
which the two .alpha.-helical peptides are aligned such that they
have the same orientation such that the amino-terminal end of one
helix is aligned with the amino-terminal end of the second
.alpha.-helix, and vice versa. Thus, the relative orientation of
the heptad `a-g` positions of two interacting .alpha.-helical
peptides aligned in parallel orientation is in the same direction.
For instance, if the heptad positions of a first helix is defined
as `abcdefg` from the amino-terminus to the carboxyl-terminus, the
heptad positions of a second helix in a parallel orientation would
be also defined as `abcdefg` as read from the amino- to the
carboxyl-terminus.
The terms "linker", "turn", "linker sequence" or "turn sequence"
are used interchangeably herein and refer to an amino acid sequence
fragment that is part of the contiguous amino acid sequence of an
HTH polypeptide, and covalently links the two .alpha.-helical
peptide sequences of that polypeptide.
As used herein, the term "single-chain" refers to the HTH scaffold
of the present disclosure, wherein the stabilizing coiled-coil
structure is formed from different regions of a contiguous amino
acid sequence of an HTH polypeptide chain folded back in an
appropriate manner.
The term "solvent-oriented" or "solvent-exposed" refers to the
region of an entity which is directly exposed or which comes
directly into contact with the solvent in the environment or the
milieu in which it is present. In the context of the present
disclosure it is the .alpha.-helix or a .alpha.-helical part of an
HTH scaffold which is directly exposed or which comes directly into
contact with the solvent in the environment or the milieu in which
it is present. More particularly, in the context of a binding site,
where one or more amino acids located in a solvent-oriented part of
the HTH scaffold contribute to the binding site, the binding site
is considered to be formed by a solvent-oriented part of the HTH
scaffold.
A ".alpha.-helical part" of a polypeptide refers to a part of a
polypeptide of the present disclosure that has an .alpha.-helical
secondary structure.
The "hydrophobic core" of an HTH scaffold refers to the part on an
HTH scaffold which is not directly exposed to the solvent in which
it is present.
As used herein, a polypeptide of the present disclosure "binds
specifically to", "specifically binds to", is "specific to/for" or
"specifically recognizes" an antigen if such polypeptide is able to
discriminate between such antigen and one or more reference
antigen(s), since binding specificity is not an absolute, but a
relative property. The reference antigen(s) may be one or more
closely related antigen(s), which are used as reference points. For
example, specific binding can be determined with a standard ELISA
assay. Alternative methods comprise, but are not limited to Western
blots, ELISA-, RIA-, ECL-, IRMA-tests and peptide scans. The
scoring may be carried out by standard color development (e.g.
detection antibody with horseradish peroxide and tetramethyl
benzidine with hydrogen peroxide). The reaction in certain wells is
scored by the optical density, for example, at 450 nm. Typical
background (=negative reaction) may be 0.1 OD; typical positive
reaction may be 1 OD. This means the difference positive/negative
can be more than 10-fold. Typically, determination of binding
specificity is performed by using not a single reference antigen,
but a set of about three to five unrelated antigens, such as milk
powder, BSA, transferrin or the like. Additionally, "specific
binding" may relate to the ability to discriminate between
different parts of its target antigen, e.g. different domains or
regions of said target antigen, or between one or more key amino
acid residues or stretches of amino acid residues of a target
antigen.
The "affinity" of a polypeptide is represented by the equilibrium
constant for the dissociation of the polypeptide and the target
protein of interest to which it binds. The lower the K.sub.D value,
the stronger the binding strength between the said polypeptide and
the target protein of interest to which it binds. Alternatively,
the affinity can also be expressed in terms of the affinity
constant (K.sub.A), which corresponds to 1/K.sub.D. The binding
affinity of a polypeptide can be determined in a manner known to
the skilled person, depending on the specific target protein of
interest. It is generally known in the art that the K.sub.D can be
expressed as the ratio of the dissociation rate constant of a
complex, denoted as k.sub.Off (expressed in seconds.sup.-1 or
s.sup.-1), to the rate constant of its association, denoted
k.sub.On (expressed in molar.sup.-1 seconds.sup.-1 or M.sup.-1
s.sup.-1). A K.sub.D value greater than about 1 millimolar is
considered to indicate non-binding or non-specific binding.
The terms "diversified amino acid residue position" or "variant
amino acid residue position" refer to an amino acid residue
position at which at least two different amino acid residues may be
present.
As used herein, the terms "inhibiting", "reducing" and/or
"preventing" refer to a polypeptide according to the present
disclosure that specifically binds to a target protein of interest
and inhibits, reduces and/or prevents the interaction between that
target protein of interest, and its natural binding partner and/or
inhibits, reduces and/or prevents a biological activity of that
target protein of interest. The inhibiting or antagonizing activity
of a polypeptide of the present disclosure may be reversible or
irreversible, but for pharmaceutical and pharmacological
applications will typically occur reversibly. The inhibiting or
antagonizing activity of a polypeptide of the present disclosure
may be measured using a suitable in vitro, cellular or in vivo
assay.
The term "synthetic" describes a molecule that is made outside of
the human body by synthesis or synthesized, e.g. DNA. The term
"synthetic" also describes a protein, e.g. antibody or fragment
that is translated from a synthetic DNA molecule.
"Linear" as used in the present disclosure refers to a stretch of
amino acids or a (poly)-peptide that does not include any secondary
or tertiary circular structure.
The term "isolated" refers to a compound which can be e.g. a
polypeptide of the disclosure or an antigen binding moiety that is
substantially free of other polypeptides or antigen binding
moieties having different antigenic specificities. Moreover, an
isolated polypeptide or antigen binding moiety may be substantially
free of other cellular material and/or chemicals.
"Constrained" as used in the present disclosure refers to a peptide
in which the three-dimensional structure is maintained
substantially in one spatial arrangement over time. The
polypeptides within the present disclosure have a constrained
conformation. Methods of determining whether peptides are
constrained are known in the art.
"Member" as used in the present disclosure refers to one molecule
forming part of a library. In the context of the present
disclosure, this term refers to one polypeptide which is part of
the polypeptide library.
"Mixture" as used in the present disclosure refers to a solution
which contains more than a molecule and in which at least two
molecules are different. This term is particularly used in order to
describe the amino acid composition at a given position or to
describe the codons encoding the respective codons for a given
position. For example, each selected codon has a certain
probability of occurring at a diversified position. E.g., if Var1
represents an "equal mixture" of the naturally occurring amino
acids, then each of the 20 naturally occurring amino acids has the
same probability of occurring at that position, i.e. 5%.
As used herein, amino acid residues will be indicated either by
their full name or according to the standard three-letter or
one-letter amino acid code. "Natural occurring amino acids" means
the following amino acids:
TABLE-US-00002 TABLE 1 Amino acids Amino acid Three letter code One
letter code Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic
acid Asp D Cysteine Cys C Glutamic acid Glu E glutamine Gln Q
Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine
Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser
S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V
"Hydrophobic amino acid residue" refers to an amino acid or residue
having a side chain exhibiting a hydrophobicity of greater than
zero according to the normalized consensus hydrophobicity scale of
Eisenberg et al. (1984, J. Mol. Biol. 179:125-142). Genetically
encoded hydrophobic amino acids include P, I, F, V, L, W, M, A and
Y.
TABLE-US-00003 TABLE 2 Normalized consensus hydrophobicity scale of
Eisenberg Eisenberg consensus scale (ECS) R K D Q N E H S T P Y C G
A M W L V F I -2.5 -1.5 -0.90 -0.85 -0.78 -0.74 -0.40 -0.18 -0.05
0.12 0.26 0.29 0.48 0.- 62 0.64 0.81 1.1 1.1 1.2 1.4
The term "vector" refers to a polynucleotide molecule capable of
transporting another polynucleotide to which it has been linked.
Preferred vectors are those capable of autonomous replication
and/or expression of nucleic acids to which they are linked. One
type of vector is a "plasmid", which refers to a circular double
stranded DNA loop into which additional DNA segments may be
ligated. Another type of vector is a viral vector, wherein
additional DNA segments may be ligated into the viral genome.
Certain vectors are capable of autonomous replication in a host
cell into which they are introduced (e.g., bacterial vectors having
a bacterial origin of replication and mammalian vectors). Other
vectors can be integrated into the genome of a host cell upon
introduction into the host cell, and thereby are replicated along
with the host genome. Vectors may be compatible with prokaryotic or
eukaryotic cells. Prokaryotic vectors typically include a
prokaryotic replicon which may include a prokaryotic promoter
capable of directing the expression (transcription and translation)
of the peptide in a bacterial host cell, such as Escherichia coli
transformed therewith. A promoter is an expression control element
formed by a DNA sequence that permits binding of RNA polymerase and
transcription to occur. Promoter sequences compatible with
bacterial hosts are typically provided in plasmid vectors
containing convenience restriction sites for insertion of a DNA
segment. Examples of such vector plasmids include pUC8, pUC9,
pBR322, and pBR329, pPL and pKK223, available commercially.
"Expression vectors" are those vectors capable of directing the
expression of nucleic acids to which they are operatively linked
and is intended to include such other forms of expression vectors,
such as viral vectors (e.g., replication defective retroviruses,
adenoviruses and adeno-associated viruses), which serve equivalent
functions.
"Display vector" includes a DNA sequence having the ability to
direct replication and maintenance of the recombinant DNA molecule
extra chromosomally in a host cell, such as a bacterial host cell,
transformed therewith. Such DNA sequences are well known in the
art. Display vectors can for example be phage vectors or phagemid
vectors originating from the class of fd, M13, or fl filamentous
bacteriophage. Such vectors are capable of facilitating the display
of a protein including, for example, a binding protein or a
fragment thereof, on the surface of a filamentous bacteriophage.
Display vectors suitable for display on phage, ribosomes, DNA,
bacterial cells or eukaryotic cells, for example yeast or mammalian
cells are also known in the art, for example, as are viral vectors
or vectors encoding chimeric proteins.
The term "recombinant host cell" (or simply "host cell") refers to
a cell into which a recombinant expression vector has been
introduced. It should be understood that such terms are intended to
refer not only to the particular subject cell but to the progeny of
such a cell. Because certain modifications may occur in succeeding
generations due to either mutation or environmental influences,
such progeny may not, in fact, be identical to the parent cell, but
are still included within the scope of the term "host cell" as used
herein. Typical host cells are prokaryotic (such as bacterial,
including but not limited to E. coli) or eukaryotic (which includes
yeast, mammalian cells, and more). Bacterial cells are preferred
prokaryotic host cells and typically are a strain of Escherichia
coli (E. coli) such as, for example, the E. coli strain DH5
available from Bethesda Research Laboratories, Inc., Bethesda, Md.
Preferred eukaryotic host cells include yeast and mammalian cells
including murine and rodents, preferably vertebrate cells such as
those from a mouse, rat, monkey or human cell line, for example
HKB11 cells, PERC.6 cells, or CHO cells.
The term "epitope" refers to an antigenic determinant, i.e. the
part of an antigen that is recognized by a binding molecule, such
as an antibody or a peptide.
The terms "binding region", "binding site" and "interaction site"
as used herein refer to a particular site, part, domain or stretch
of amino acid residues present on the polypeptides of the present
disclosure that is responsible for binding to a target molecule.
Such binding region consists of specific amino acid residues from
the said polypeptide which are in contact with the target
molecule.
"Phage display" is a technique by which variant polypeptides are
displayed as fusion proteins to a coat protein on the surface of
phage, e g filamentous phage particles, while the genetic material
encoding each variant resides on the inside. This creates a
physical linkage between each variant protein sequence and the DNA
encoding it, which allows rapid partitioning based on binding
affinity to a given target molecule (antibodies, enzymes,
cell-surface receptors, etc.) by an in vitro selection process
called panning. In its simplest form, panning is carried out by
incubating a library of phage-displayed peptides on a plate (or
bead) coated with the target, washing away the unbound phage, and
eluting the specifically bound phage. The eluted phage are then
amplified and taken through additional binding/amplification cycles
to enrich the pool in favor of binding sequences. After a few
rounds, individual clones are characterized by DNA sequencing and
ELISA.
A utility of phage display lies in the fact that large libraries of
randomized protein variants can be rapidly and efficiently sorted
for those sequences that bind to a target molecule with high
affinity display of peptides and proteins libraries on phage has
been used for screening millions of polypeptides for ones with
specific binding properties. Polyvalent phage display methods have
been used for displaying small random peptides and small proteins
through fusions to either gene III or gene VIII of filamentous
phage (Wells and Lowman ((1992) Curr Opin Struct Biol B 355-362)
and references cited therein). In monovalent phage display, a
protein or peptide library is linked to a gene III or a portion
thereof and expressed at low levels in the presence of wild type
gene III protein so that phage particles display one copy or none
of the fusion proteins.
The term "phage vector" means a double stranded replicative form of
a bacteriophage containing a heterologous gene and capable of
replication. The phage vector has a phage origin of replication
allowing phage replication and phage particle formation. The phage
is preferably a filamentous bacteriophage, such as, an M13, fd
phage or a derivative thereof, a lambdoid phage, such as lambda, a
Baculovirus, a T4 phage, a T7 phage virus, or a derivative of any
of the foregoing.
The term "coat protein" means a protein, at least a portion of
which is present on the surface of the virus particle. From a
functional perspective, a coat protein is any protein which
associates with a virus particle during the viral assembly process
in a host cell, and remains associated with the assembled virus
until it infects another host cell. The coat protein may be the
major coat protein, such as pVIII, or may be a minor coat protein,
such as pIII.
The term "antibody" as used herein refers to a protein comprising
at least two heavy (H) chains and two light (L) chains
inter-connected by disulfide bonds which interacts with an antigen.
Each heavy chain is comprised of a heavy chain variable region
(abbreviated herein as VH) and a heavy chain constant region. The
heavy chain constant region is comprised of three domains, CH1, CH2
and CH3. Each light chain is comprised of a light chain variable
region (abbreviated herein as VL) and a light chain constant
region. The light chain constant region is comprised of one domain,
CL. The VH and VL regions can be further subdivided into regions of
hypervariability, termed complementarity determining regions (CDR),
interspersed with regions that are more conserved, termed framework
regions (FR). Each VH and VL is composed of three CDRs and four
FR's arranged from amino-terminus to carboxy-terminus in the
following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The
variable regions of the heavy and light chains contain a binding
domain that interacts with an antigen. The constant regions of the
antibodies may mediate the binding of the immunoglobulin to host
tissues or factors, including various cells of the immune system
(e.g., effector cells) and the first component (Clq) of the
classical complement system. The term "antibody" includes for
example, monoclonal antibodies, human antibodies, humanized
antibodies, camelised antibodies and chimeric antibodies. The
antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA and
IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or
subclass. Both the light and heavy chains are divided into regions
of structural and functional homology.
The phrase "antibody fragment", as used herein, refers to one or
more portions of an antibody that retain the ability to
specifically interact with (e.g., by binding, steric hindrance,
stabilizing spatial distribution) an antigen. Examples of binding
fragments include, but are not limited to, a Fab fragment, a
monovalent fragment consisting of the VL, VH, CL and CH1 domains; a
F(ab)2 fragment, a bivalent fragment comprising two Fab fragments
linked by a disulfide bridge at the hinge region; a Fd fragment
consisting of the VH and CH1 domains; a Fv fragment consisting of
the VL and VH domains of a single arm of an antibody; a dAb
fragment (Ward et al., (1989) Nature 341:544-546), which consists
of a VH domain; and an isolated complementarity determining region
(CDR). Furthermore, although the two domains of the Fv fragment, VL
and VH, are coded for by separate genes, they can be joined, using
recombinant methods, by a synthetic linker that enables them to be
made as a single protein chain in which the VL and VH regions pair
to form monovalent molecules (known as single chain Fv (scFv); see
e.g., Bird et al., (1988) Science 242:423-426; and Huston et al.,
(1988) Proc. Natl. Acad. Sci. 85:5879-5883). Such single chain
antibodies are also intended to be encompassed within the term
"antibody fragment". These antibody fragments are obtained using
conventional techniques known to those of skill in the art, and the
fragments are screened for utility in the same manner as are intact
antibodies. Antibody fragments can also be incorporated into single
domain antibodies, maxibodies, minibodies, intrabodies, diabodies,
triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger
and Hudson, (2005) Nature Biotechnology 23:1126-1136). Antibody
fragments can be grafted into scaffolds based on polypeptides such
as Fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which
describes fibronectin polypeptide monobodies). Antibody fragments
can be incorporated into molecules comprising a pair of tandem Fv
segments (VH--CH1-VH--CH1) which, together with complementary light
chain polypeptides, form a pair of antigen-binding sites (Zapata et
al., (1995) Protein Eng. 8:1057-1062; and U.S. Pat. No.
5,641,870).
DETAILED DESCRIPTION OF THE INVENTION
The present disclosure provides novel polypeptide libraries that
are conformationally constrained in an anti-parallel,
helix-turn-helix (HTH) arrangement. The present disclosure further
relates to methods of generating and screening such libraries to
identify polypeptides for biological, pharmaceutical and other
uses.
The polypeptide library of the present disclosure can be used for
the screening and/or selection of one or more polypeptides that
specifically bind to a target molecule of interest.
It has been found that polypeptides isolated from the library have
certain preferred properties and are superior over other binding
agents known in the art. Such properties include target binding
with high affinities, a compact and small size (.about.6 kDa), low
immunogenicity, extreme stability against thermal and chemical
denaturation, and insensitiveness to changes in pH and to
proteolytic degradation.
Design of the Library
In order to design a scaffold structure suitable for a structurally
constraint presentation of the various sequence combinations, a
novel and unique approach in combining statistical and structural
amino acid propensities occurring in natural .alpha.-helices was
used.
The most abundant amino acid residues found in natural
.alpha.-helices may have favorable biophysical properties that lead
to more efficient development and increase the safety and efficacy
of the resulting polypeptides in patients. Such favorable
biophysical properties include high relative display rate, high
expression yields, low immunogenicity, resistance against
temperature and chemical denaturation, relative insensitiveness for
pH alterations, serum stability and resistance against proteolytic
degradation by proteases.
Data about the abundancy of natural occurring amino acids in
.alpha.-helices can be obtained from publically available
literature, such as Aurora et al. (Protein Science (1998), 7:21-38)
and Pace et al. (Biophysical Journal (1998), Vol. 75, 422-427).
These data can be compiled to catalog amino acid preferences at
certain positions of the .alpha.-helical coiled-coil structure in
an aqueous solution. By this approach a design template can be
generated, e.g. a design template for a .alpha.-helical reference
sequence consisting of 15 amino acid residues, consisting of two
consecutive heptad sequences.
For each of said 15 amino acid residue positions, the five to six
most frequently occurring amino acids residues are considered as a
basis for designing two independent .alpha.-helical peptide
sequences, named Helix-1 and Helix-2, respectively.
In order to select the optimal reference sequence for the
generation of a polypeptide library according of the present
disclosure, additional structural consideration for promoting helix
formation and stabilizing the .alpha.-helical coiled-coil structure
were taken into account to select the most appropriate amino
residue at each position.
Accordingly, a reference polypeptide sequence (HTHdes2) comprising
two .alpha.-helical peptides and a linker segment was designed.
Testing via circular dichroism (CD) spectra measurements confirmed
that the HTHdes2 reference sequence resulted in a high degree of
.alpha.-helical content and a low content of random coiled
structures in solution. Additional studies revealed that the
HTHdes2 sequence is extreme resistant towards thermal and chemical
denaturation.
The polypeptide library of the present disclosure is based on a
helix-turn-helix (HTH) scaffold structure which comprises two
.alpha.-helical peptides which are orientated in an anti-parallel
arrangement, such that said .alpha.-helical peptides are capable of
forming a stabilized coiled-coil structure. The two .alpha.-helical
peptides constituting the HTH scaffold of the present disclosure
are herein referred to as Helix-1 and Helix-2.
Accordingly, Helix-1 and Helix-2 assemble into a helix-turn-helix
scaffold in an anti-parallel configuration, wherein the two helices
are arranged such that the amino-terminal end of Helix-1 is aligned
with the carboxyl-terminal end of Helix-2.
In certain embodiments of the disclosure, the two .alpha.-helical
peptides, Helix-1 and Helix-2, are of similar size, each ranging
from about 10 to about 50 residues in length. In another
embodiment, Helix-1 and Helix-2 are of equal length. In another
embodiment, Helix-1 and Helix-2 are 15 amino acid residues in
length.
The assembly of Helix-1 and Helix-2 occurs due to the presence of a
repeated heptad motif of conserved amino acid residues.
In a preferred embodiment of the present disclosure, Helix-1 and
Helix-2 are formed by a single polypeptide wherein the two
.alpha.-helical peptides are either directly linked via a single
peptide bond to each other, or are linked by a linker segment that
does not substantially interfere with the association of Helix-1
and Helix-2 into a coiled-coil structure.
In an embodiment of the present disclosure, the two .alpha.-helical
peptides are covalently linked by a flexible linker (Li) in a way
that the carboxyl-terminus of the first .alpha.-helical peptide
(Helix-1) is linked to the amino-terminus of the second
.alpha.-helical peptide (Helix-2).
Thus, according to a specific embodiment of the present disclosure,
the HTH scaffold structure of the library of the present disclosure
comprises the general formula Helix-1-Li-Helix-2.
Each of the scaffold structure forming .alpha.-helical peptides is
comprised of a peptide whose sequence contains "invariant"
positions, i.e. positions which contain the same amino acid
residues in each member of the library, and "variable" positions,
i.e. positions which contain different amino acid residues in the
different members of the library. These variable positions are
important to diversify the library, i.e. to generate a library
consisting of different members.
In an embodiment of the present disclosure, the introduced sequence
variation or variable positions within the library is present on
both .alpha.-helical peptides, which form the HTH scaffold of the
present disclosure.
In an embodiment of the present disclosure, Helix-1 and Helix-2 may
have the same amino acid residues at their invariant positions.
In another embodiment of the present disclosure, Helix-1 and
Helix-2 have different amino acid residues at their invariant
positions.
In particular embodiments of the present disclosure, the invariant
positions of Helix-1 and Helix-2 do not correspond to a naturally
occurring protein sequence. In another embodiment of the present
disclosure, the invariant positions of Helix-1 and Helix-2 are of
non-natural origin. In another embodiment of the present
disclosure, the invariant positions of Helix-1 and Helix-2 is are
artificial sequences.
In an aspect of the present disclosure, the amino acid residues
present at the invariant positions of Helix-1 and Helix-2 are
referred to as X1, X2, X3, X4, X5, and Hy, respectively (see FIG.
1).
The invariant amino acid residues X1, X2, X3, X4, and X5 are
solvent exposed since they are located at the outward-facing side
of the HTH scaffold, and are in contact with the solvent when the
HTH scaffold structure is in solution.
In an embodiment of the present disclosure, the invariant amino
acid residue X1 is selected from the group of D, T, N, S and P. In
another embodiment of the present disclosure, the invariant amino
acid residue X1 is D in Helix-1 and D in Helix-2.
In an embodiment of the present disclosure, the invariant amino
acid residue X2 is selected from the group of E, P, Q, W and D. In
another embodiment of the present disclosure, the invariant amino
acid residue X2 is E in Helix-1 and E in Helix-2.
In an embodiment of the present disclosure, the invariant amino
acid residue X3 is selected from the group of M, A, I, Q and R. In
another embodiment of the present disclosure, the invariant amino
acid residue X3 is Q in Helix-1 and A in Helix-2.
In an embodiment of the present disclosure, the invariant amino
acid residue X4 is selected from the group of A, L, R, M, K and E.
In another embodiment of the present disclosure, the invariant
amino acid residue X4 is E in Helix-1 and K in Helix-2.
In an embodiment of the present disclosure, the invariant amino
acid residue X5 is selected from the group of M, L, A, W, F and K.
In another embodiment of the present disclosure, the invariant
amino acid residue X5 is K in Helix-1 and M in Helix-2.
The amino acid residues that are varied in the polypeptide library
of the present disclosure and that contribute to diversity are
referred to as Var1 Var2, and Var3. This corresponds to heptad
positions b, e, and f of the .alpha.-helical peptides Helix-1 and
Helix-2.
Helix-1 and Helix-2 reversibly bind to one another in a manner that
is determined by the identity of the residues at the invariant
positions of the two .alpha.-helical peptides Helix-1 and
Helix-2.
Helix-1 and Helix-2 of to the present disclosure are each comprised
of two "heptads" and thus may be referred to as a "heptad repeats".
The heptad repeats give rise to regularly repeating heptad
positions, corresponding to regularly-repeating amino acid residues
along the .alpha.-helix (FIGS. 1 and 3).
The relative orientations of the `a-g` positions of the two
interacting .alpha.-helices arranged in an anti-parallel
configuration of the present disclosure is shown in FIGS. 1 and
3.
In an aspect of the present disclosure, the HTH scaffold is
stabilized primarily by non-covalent bonds. In preferred
embodiments, said non-covalent bonds are formed by hydrophobic
interactions between hydrophobic residues at the contact region
between Helix-1 and Helix-2.
Accordingly, the individual .alpha.-helical peptides Helix-1 and
Helix-2 contact one another along their respective hydrophobic
faces, formed by the regularly repeating amino acid residues Hy.
This corresponds to heptad positions `a` and `d`.
The contact region of Helix-1 and Helix-2 comprises the hydrophobic
core of the helix-turn-helix scaffold of the present disclosure. In
one aspect, said hydrophobic amino acids residues are invariant
amino acid residues.
The appropriate selection of the hydrophobic residues Hy at the
heptad positions `a` and `d` position is important for the
formation of a coiled-coil structure.
In an embodiment of the present disclosure, said hydrophobic amino
acids have a hydrophobicity of greater 0.62 according to the
normalized consensus hydrophobicity scale of Eisenberg et al.
(1984, J. Mol. Biol. 179:125-142).
In an embodiment of the present disclosure, Hy is an hydrophobic
amino acid selected from the group, such as I, F, V, L, W, M. In
another embodiment of the present disclosure, Hy is selected from
the group of I, L, and V. In yet another embodiment of the present
disclosure, the hydrophobic amino acid Hy is L.
In further embodiments, the HTH scaffold of the present disclosure
may be further stabilized by the introduction of negatively charged
amino acid residues at the amino-terminal end of each
.alpha.-helical peptide. This may stabilizes the dipole moment of
the .alpha.-helices of said peptides. Such a negatively charged
amino acid residue can be D or E. In another embodiment, such a
negatively charged amino acid residue is D.
In further embodiments, the HTH scaffold of the present disclosure
may further be stabilized by inter- or intrahelical electrostatical
interactions. Such electrostatical interactions may be ionic
interactions.
In further embodiments, the HTH scaffold of the present disclosure
is stabilized by intrahelical ionic interactions between the
invariant amino acid residues
X2 of Helix-1 and X4 of Helix-2,
X4 of Helix-1 and X4 of Helix-2, and/or
X5 of Helix-1 and X2 of Helix 2.
Preferably, X2 and X4 of Helix-1 are negatively charged amino acid
residues and X5 is a positively charged amino acid residue.
Negatively charged amino acid residue may be D or E, positively
charged amino acid residue may be K, H or R.
Preferably, X2 of Helix-2 is a negatively charged amino acid
residue and X4 of Helix-2 is a positively charged amino acid
residue.
In further embodiments, the HTH scaffold of the present disclosure
is stabilized by interhelical ionic interactions between the
invariant amino acid residues X4 and X5 of Helix-1. Preferably, X4
is a negatively charged amino acid residue and X5 is a positively
charged amino acid residue. In another embodiment, X4 is E and X5
is K.
In further embodiments, the HTH scaffold of the present disclosure
is stabilized by interhelical hydrogen bonding.
In one embodiment, said interhelical hydrogen bonding is between
the invariant amino acid residues X2 and X3 and/or between X3 and
X4 of Helix-1. In another embodiment, X2 is E, X3 is Q and X4 is
E.
Linker
The two .alpha.-helical peptides (Helix-1 and Helix-2) of the
present disclosure are linked via a peptide linker (Li) which
connects the carboxyl-terminus of Helix-1 to the amino terminus of
Helix-2 thus resulting in a single-chain amino acid sequence for
the polypeptides of the disclosure.
In an embodiment, Helix-1, Li and Helix-2 are covalently linked in
a way that the carboxyl-terminus of Helix-1 is linked to the
amino-terminus of Li and that the carboxyl-terminus of Li is linked
to the amino-terminus of Helix-2. In another embodiment, the
arrangement from the amino- to the carboxyl-terminus is as follows:
Helix-1, Li and Helix-2. A typical Helix 1-Linker-Helix 2 structure
according to the present disclosure is depicted in FIGS. 1 and
3.
Preferably, the linker comprises a non-helical region. The first
and last residues of the non-helical region can be any amino acid.
Preferably, one or both residues are helix breaking or helix
destabilizing residues, such as glycine or proline.
Such peptide linker include for example, but are not limited to,
glycine linkers, serine linkers, mixed glycine/serine linkers,
glycine- and serine-rich linkers, which are known to the skilled
artisan.
In an embodiment of the present disclosure, the linker is
absent.
In an embodiment of the present disclosure, the linker has a length
of 1 to 50 amino acid residues. In another embodiment, the linker
has a length of 1-30 amino acid residues. Yet in another
embodiment, the linker has a length of 1-10 amino acid residues. In
one embodiment, the linker has a length of 5 amino acids. In one
embodiment, the linker comprises the amino acid sequence QGVDS (SEQ
ID NO: 6). In one embodiment, the linker has the amino acid
sequence QGVDS (SEQ ID NO: 6).
Variability of the Library
In general, a coiled-coil motif is tolerant to amino acid
substitutions provided that the .alpha.-helix is not substantially
destabilized.
The polypeptide library of the present disclosure is characterized
by defined diversified positions at which mixtures of amino acid
residues are placed. The diversified positions within the library
is present on both .alpha.-helical peptides (Helix-1 and Helix-2)
forming the HTH scaffold structure of the present disclosure.
In an embodiment, 6 positions in Helix-1 and 6 positions in Helix-2
are diversified.
Accordingly, the amino acid variations at the variable positions of
the HTH scaffold of the present disclosure give rise to polypeptide
members with different sequences.
The amino acid residues at variant positions are referred to as
Var1, Var2, and Var3. This corresponds to heptad positions `b`,
`e`, `f` within the two .alpha.-helical peptides of the HTH
scaffold.
The diversified amino acid residue positions are located on one
face of the solvent exposed regions of the HTH scaffold, thereby
forming the predominant binding site on the polypeptide members of
the library.
As can be seen in FIGS. 2A and 2B, Var1 represents solvent-exposed
amino acid residues which align on the same side of the HTH
scaffold of the present disclosure and thus were used for
diversification to generate the library of the present disclosure.
See also Example 1.
In order to further increase the library size and to increase the
contact interphase between the polypeptides of the library and
their bound target molecules of interest, additional variable
positions were introduced in Helix-1 and Helix-2. As denoted above,
the amino acid residues at the additional variant positions of the
present disclosure are referred to as Var2 and Var3,
respectively.
The diversification employed in the library of the present
disclosure may encompass both, naturally occurring and synthetic
amino acid residues.
However, in certain embodiments, the diversified amino acid residue
Var1, Var2 and Var3 are exclusively occupied by a mixture of the
naturally occurring amino acids, as defined herein.
In a preferred embodiment of the present disclosure, said mixture
is an equal mixture of the naturally occurring amino acids.
In yet a further embodiment, the diversified amino acid residue
Var1 comprises a mixture of naturally occurring amino acids
excluding G, P, C. G, P, C are known to break .alpha.-helical
structures and thus were avoided. C was furthermore excluded in
order to avoid the formation of potential di-sulfide bonds between
two or more diversified amino acid residue positions. Furthermore,
it was shown, that C residues appear significant less frequent in
natural occurring .alpha.-helices compared to other natural
occurring amino acid residue (Aurora et al., Protein Science
(1998), 7:21-38) and Pace et al. (Biophysical Journal (1998), Vol.
75, 422-427)).
In an embodiment of the present disclosure, the diversified amino
acid residue positions Var2 and Var3 comprise the naturally
occurring amino acid residues E, D, K, R, N, Q, and H. In a
preferred embodiment, said mixture is an equal mixture of said
amino acid residues.
In one embodiment, Var2 comprises a mixture of the naturally
occurring amino acid residues R, E, Q and Var3 comprises a mixture
of the naturally occurring amino acid residues R, Q, H. In a
preferred embodiment, said mixture is an equal mixture.
The library members of the present disclosure are characterized in
that said polypeptides differ from each other in the defined set of
12 diversified amino acid residue positions.
Accordingly, the library members can differ from each other in at
least one amino acid residue positions within the defined set of 12
diversified amino acid residue positions. Alternatively, the
library members can differ from each other in 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, or all 12, of these amino acid residue positions.
Accordingly, the library members can be distinguished from each
other by the sequence difference(s) present in the defined set of
the 12 diversified amino acid residue positions.
In certain embodiments, the polypeptide library of the present
disclosure is displayed on bacteriophage. Phage display is known to
have significant advantages in allowing the rapid selection of
useful molecules. This method allows the preparation of libraries
as large as 10.sup.10 unique peptide members, many orders of
magnitude larger than libraries that may be prepared synthetically.
Using such a robust platform allows for the display of large,
diverse libraries.
In certain embodiments, a library of polypeptides according the
present disclosure contains at least 10.sup.2, at least 10.sup.3,
at least 10.sup.4, at least 10.sup.5, at least 10.sup.6, at least
10.sup.7, at least 10.sup.8, at least 10.sup.9 or even more
different library members. In one embodiments, a library or
collection of polypeptides of the present disclosure contains at
least 10.sup.6 different library members.
In certain embodiments, the present disclosure provides a
collection of nucleic acids encoding the library of polypeptides of
the present disclosure.
In certain embodiments, the present disclosure provides a vector
comprising the collection of nucleic acids encoding the library of
polypeptides of the present disclosure.
In certain embodiments, the vector is a display vector. In other
embodiments, the vector is an expression vector.
In certain embodiments, the present disclosure provides a
recombinant host cell comprising the collection of nucleic acid
molecules or the vector encoding the library of polypeptides of the
present disclosure.
In certain embodiments, the present disclosure provides a method to
isolate a polypeptide specific for an antigen, said method
comprising the steps of:
a) contacting the library of the present disclosure with an
antigen,
b) removing those members of the library which do not bind to the
antigen; and
c) recovering those members of the library bound to the antigen
In certain embodiments, the present disclosure provides a
polypeptide identified using the library of polypeptides disclosed
herein.
In an embodiment, the present disclosure provides a
helix-turn-helix scaffold structure to an antibody or an antibody
fragment, wherein said helix-turn-helix scaffold structure
comprises a first and a second .alpha.-helical peptide that form an
anti-parallel, coiled-coil structure.
In one aspect, the present disclosure provides a library of
polypeptides, wherein each member of the library comprises a
helix-turn-helix scaffold structure of the formula
Helix-1-Li-Helix-2, wherein Helix-1 and Helix-2 comprise a first
and second .alpha.-helical peptide, wherein each of said
.alpha.-helical peptides comprises the amino acid sequence
X1-X2-Hy-Var1-X3-Hy-Var1-Var2-X4-Hy-Var1-X5-Hy-Var1-Var3 (SEQ ID
NO: 1), wherein X1 is D, T, N, S or P, X2 is E, P, Q, W or D, X3 is
M, A, I, Q or R, X4 is A, L, R, M, K or E, X5 is M, L, A, W, F or
K, Hy is any amino-acid residue having a side chain exhibiting a
hydrophobicity of greater than 0.62, and Var1, Var2 and Var3 are
mixtures of the natural occurring amino acids, excluding G, P, and
C,
Li is a linker, and
said first and second .alpha.-helical peptide form an
anti-parallel, coiled-coil structure.
In one embodiments of the present disclosure, additional amino acid
residues at the amino terminus and/or the carboxyl-terminus of the
helix-turn-helix scaffold are added.
Amino acid residues may also be replaced, deleted or added, for
example to aid in the expression of library members in a preferred
host species, to facilitate cloning of the molecule, to increase
the stability of the peptide; to increase helix packing and so on.
In one embodiment of the present disclosure, G is introduced at the
amino- and carboxyl-terminus of the helix-turn-helix scaffold.
Methods for Generating Diversified Gene Libraries
Numerous methods for the generation of diversified genes and gene
libraries are known. This includes the Slonomics technology (U.S.
Ser. No. 12/414,174 and Van den Brulle et al., Biotechniques
(2008), 45, 340-343)).
The Slonomics technology uses a defined number of standardized
building blocks containing self-complementary regions. Two
different classes of building blocks (called "splinkers" and
"anchors") are used to successively build up tailor-made nucleotide
libraries with any desired bias.
The diversified trinucleotide-containing oligonucleotide (TRIM)
technology (WO93/21203) as well as oligonucleotide-mediated
mutagenesis (Zoller et al. ((1987) Nucleic Acids. Res. 10
6487-6504)) are further methods for preparing diversified gene
libraries.
Cassette mutagenesis is a further method for preparing the
diversified gene libraries. The method is based on that described
by Wells et al. ((1985) Gene 34:315).
Diversified gene libraries may be also prepared by standard solid
phase peptide synthesis (Merrifield et al., J. Am. Chem. Soc.,
1963, 85 (14), pp 2149-2154) with subsequent mixing of the purified
products.
In certain embodiments, the synthesis of the polypeptide library of
the present disclosure involves the step of producing a nucleic
acid or vector library of at least 100 members, wherein each member
encodes a polypeptide according to the present disclosure, and
wherein the encoded different library members differ from each
other in at least one out of a defined set of 12 diversified amino
acid residue positions.
Upon expression in host cells, the polypeptide library of the
present disclosure is obtained. These steps can be achieved in
different ways, as will be known by the person skilled in the art.
In general, such steps typically include transforming or
transfecting a suitable host cell with a nucleic acid or vector
library or an infectious particle which encodes the polypeptide
library. Further, such steps typically include culturing said host
cells under conditions suitable for the proliferation
(multiplication, growth) of said host cells and a culturing step
under conditions suitable for the production (expression,
synthesis) of the encoded polypeptides. The culturing of host cells
under conditions suitable for proliferation or expression is
typically accomplished in the presence of media comprising
components suitable for cell growth or induction of expression. In
particular embodiments, the methods for the production of
polypeptide libraries of the present disclosure further comprise
the step of isolating the produced polypeptide from the host cells
or medium. It is further noted that the expressed polypeptide
libraries may, in addition to the different-sequence polypeptides,
also contain multiple copies of identical polypeptides.
Phage-Display Methods
Phage display methods for proteins, peptides and mutated variants
thereof, including constructing a family of variant replicable
vectors containing a transcription regulatory element operably
linked to a gene fusion encoding a fusion polypeptide, transforming
suitable host cells, culturing the transformed cells to form phage
particles which display the fusion polypeptide on the surface of
the phage particle, contacting the recombinant phage particles with
a target molecule so that at least a portion of the particle bind
to the target, separating the particles which bind from those that
do not bind, are known to the skilled artisan and may be used with
the libraries disclosed herein.
In certain embodiments, the polypeptides of the present disclosure
are linked to at least a portion of a phage coat protein to form a
fusion protein containing the polypeptide disclosed herein. The
fusion protein can be made by expressing a gene fusion encoding the
fusion protein using known techniques of phage display. The fusion
protein may form part of a phage or phagemid particle in which one
or more copies of the peptide are displayed on the surface of the
particle.
In certain embodiments, the present disclosure provides vectors
comprising the fusion genes noted above, as well as a library of
these vectors. The library of vectors may be in the form of a DNA
library, a library of virus (phage or phagemid) particles
containing the library of fusion genes or in the form of a library
of host cells containing a library of the expression vectors or
virus particles.
In certain embodiments, the present disclosure provides a method
comprising the steps of preparing a library containing a plurality
of vectors, each vector comprising a transcription regulatory
element operably linked to a gene fusion encoding a fusion protein,
wherein the gene fusion comprises a first gene encoding a peptide
disclosed herein and a second gene encoding at least a portion of a
phage coat protein, wherein the library comprises a plurality of
genes encoding polypeptide fusion proteins.
The gene encoding the coat protein of the phage and the gene
encoding the desired polypeptide portion of the fusion protein of
the present disclosure (the polypeptide of the present disclosure
linked to at least a portion of a phage coat protein) can be
obtained by methods known in the art (see generally, Sambrook et
al.) The DNA encoding the gene may be chemically synthesized
(Merrfield (1963) 7 Am Chem Soc 85:2149).
The phage coat protein is preferably the gene III or gene VIII coat
protein of a filamentous phage, such as, M13. Any suitable gene III
vectors for display of peptides may be used, including fd-CATI
(McCafferty et al. (1990) Nature (London) 348 552-554) and pHEN I
(Hoogenboom et al. (1991) Nucleic Acids Res 19 4133-4137).
Suitable phage vectors, phagemid vectors and helper phage for use
in accordance with the present disclosure are known to the skilled
artisan.
Any suitable cells which can be transformed by electroporation may
be used as host cells in the method of the present disclosure.
Suitable host cells which can be transformed include gram negative
bacterial cells such as E. coli. Suitable E. coli strains may
include TG1F- or E. coli XL-I Blue (Stratagene).
In certain embodiments the host cell for electroporation is a
competent E. coli strain containing a phage F' episome. Any F'
episome which enables phage replication in the strain may be
used.
After selection of the transformed cells, these cells are grown in
culture and the vector DNA may then be isolated. Phage or phagemid
vector DNA can be isolated and analysed using methods known in the
art, for example, as described in Sambrook et al.
Panning Methods
Various panning methods may be used in accordance with the present
disclosure. In a direct panning protocol the target is immobilized
on a solid support. Examples of solid support are microtiter plates
or tubes (e.g. Maxisorp plates, Maxisorp tubes, Nunc) or magnetic
beads (Dynabeads, Invitrogen). The target can either be directly
coated on plastic or beads (e.g. surface activated beads, such as
Dynabeads), or via streptavidin when the target is biotinylated.
Other tags can be used to capture the targets such as His-tags or,
alternatively, an antibody directed against the target can also be
used to capture the target on the support.
Also solution panning protocol may be used. Here, the target is
captured on the solid support after incubation with the phage
library. The target-phage interaction is performed in solution. To
be able to wash away the non-binding phage, the target needs to be
immobilized on a solid support.
In certain embodiments, an Fc-tagged target is used, whereby
phages, which display a polypeptide of the present disclosure
binding to the target, are captured with a Protein G or Protein A
coated support (e.g. magnetic beads).
Polypeptides of the Present Disclosure
The polypeptides of the present disclosure can be synthesized by a
variety of means, for example, by recombinant DNA technology or by
chemical synthesis. Methods of peptide synthesis are known in the
art.
Alternatively, the coding sequences for the polypeptides can be
recombinant DNA molecules, which are introduced into expression
vectors or phage by operatively linking the DNA to the necessary
expression control regions (e.g. regulatory regions) required for
gene expression.
The vectors can be introduced into the appropriate host cells such
as prokaryotic (e.g., bacterial) or eukaryotic (e.g., yeast or
mammalian) cells by methods well known in the art (see, e.g.,
"Current Protocol in Molecular Biology", Ausubel et al. (eds.),
Greene Publishing Assoc. and John Wiley Interscience, New York,
1989 and 1992). Numerous cloning vectors are known to those of
skill in the art, and the selection of an appropriate cloning
vector is a matter of choice.
The gene can be placed under the control of a promoter, ribosome
binding site (for bacterial expression) and, optionally, an
operator (collectively referred to herein as "control" elements),
so that the DNA sequence encoding the desired protein is
transcribed into RNA in the host cell transformed by a vector
containing this expression construction. The coding sequence may or
may not contain a signal peptide or leader sequence.
Depending on the expression system and host selected, the proteins
of the present disclosure are produced by growing host cells
transformed by an expression vector described above under
conditions whereby the protein of interest is expressed.
The protein is then isolated from the host cells and purified. If
the expression system secretes the protein into growth media, the
protein can be purified directly from the media. If the protein is
not secreted, it is isolated from cell lysates or recovered from
the cell membrane fraction. The selection of the appropriate growth
conditions and recovery methods are within the skill of the
art.
The polypeptides of the present disclosure can then be purified by
a number of techniques as known to the person skilled in the
art.
It should be noted that the libraries and polypeptides of the
disclosure are not naturally occurring proteins. Typically, the
polypeptides of the present disclosure are recombinant, synthetic
or semi-synthetic amino acid sequences, polypeptides or
proteins.
As further described herein, the total number of amino acid
residues in a polypeptide of the present disclosure can be in the
range of 25 to 50000, in the range of 25-10000, in the range of 25
to 5000, in the range of 25-1000, in the range of 25-500, in the
range of 25-250, in the range of 25-100, in the range of 25-50, or
in the range of 25-35, depending mainly on the length of the
flexible linkers interconnecting the two .alpha.-helical peptides
and the additional moieties which may are linked to the
helix-turn-helix scaffold.
The polypeptides of the present disclosure can be synthesized with
additional amino acid residues added at their amino- and the
carboxyl-terminus in order to allow linkage of the amino- and
carboxyl terminal end of the polypeptides. The thus formed cyclized
polypeptides may further increase the stability of the
.alpha.-helical structure and improve resistance against
proteolytic degradation by proteases.
The polypeptides of the present disclosure can be cyclized,
introducing e.g. a disulfide bridge or a reduction insensitive
thioether linkage. A disulfide bonde can be formed under oxidizing
conditions between amino- and carboxyl-terminally introduced
cysteine residues. A thioether bond according to the present
disclosure may be formed for instance between N-chloroacetyl
glycine present at the amino-terminus and a C residue present at
the carboxyl-terminus.
In an embodiment of the present disclosure, the polypeptides
according to the present disclosure are cyclic polypeptides.
In an embodiment of the present disclosure, the cyclic polypeptides
are formed by a covalent bond.
In certain embodiments of the present disclosure, the covalent bond
is a disulfide bond.
In certain embodiments of the present disclosure, the disulfide
bond is formed by two C residues.
In certain embodiments of the present disclosure, the disulfide
bond is formed between a C residue present at the amino-terminal
end and a C residue present at the carboxyl-terminal end of the
polypeptides of the present disclosure.
In certain embodiments of the present disclosure, the covalent bond
is a thioether bond.
In certain embodiments of the present disclosure, the covalent bond
is a thioether bond formed between N-chloroacetyl glycine and a C
residue.
In certain embodiments, the covalent bond is a thioether bond
formed between N-chloroacetyl glycine present at the amino-terminus
and a C residues present at the carboxyl-terminus of the
polypeptides of the present disclosure.
In certain embodiments, the covalent bond is a thioether bond
formed between N-chloroacetyl glycine present at the amino-terminus
and a C residues present at the carboxyl-terminus of the
polypeptides of the present disclosure.
Polypeptide Fusion Proteins
The polypeptides provided by the library of the present disclosure
may or may not be linked to one or more other moieties.
Such fusion-proteins may be prepared in any suitable manner,
including genetically or chemically approaches.
Said linked moieties may contain secretory or leader sequences,
sequences that aid detection, expression, separation or
purification, or sequences that confer to increased protein
stability, for example, during recombinant production.
Examples of potential moieties include beta-galactosidase,
glutathione-S-transferase, luciferase, a T7 polymerase fragment, a
secretion signal peptide, an antibody or antibody fragment thereof,
a toxin, a reporter enzyme, a moiety being capable of binding a
metal ion like a poly-histidine tag, a tag suitable for detection
and/or purification, a homo- or hetero-association domain, a moiety
which increases solubility of a protein, or a moiety which
comprises an enzymatic cleavage site. Accordingly, a polypeptide of
the disclosure may optionally contain one or more moieties for
binding to other targets or target proteins of interest.
It should be clear that such further moieties may or may not
provide further functionality to the polypeptides of the disclosure
and may or may not modify the properties of the polypeptide of the
disclosure.
The polypeptides of the present disclosure may be linked to one or
more other moieties either directly by a single peptide bond or
through one or more spacer(s) containing one or more ammo acid
residues.
Suitable spacer(s) for use in linking the polypeptides of the
disclosure with one or more moieties may be any spacer region used
in the art to link peptides and/or proteins.
Some suitable spacers include for example, but are not limited to
polypeptide spacers such as glycine spacers, serine spacers, mixed
glycine/serine spacers, glycine- and serine-rich spacers, spacer
composed of largely polar polypeptide fragments or spacers
comprising an amino acid sequence forming a random coil
conformation.
A spacer may be any suitable amino acid sequence having a length
between 1 and 500 amino acid residues, such as between 1 and 100,
between 1 and 50, between 1 and 10, or between 1 and 5 amino acid
residues.
In an embodiment, the polypeptides of the present disclosure are
linked to a poly-histidine tag. In other embodiment, the
polypeptides of the present disclosure are linked to a FLAG tag.
Yet in another embodiment, the polypeptides are linked to a FLAG
and poly-histidine tag. In certain embodiments, said poly-histidine
and/or FLAG tag is linked to the carboxyl- and/or amino-terminus of
the polypeptide of the present disclosure.
In another embodiment, the polypeptides of the present disclosure
are linked to a maltose binding protein (MBP). Maltose binding
protein may increase the solubility during expression in bacterial
hosts. In certain embodiments, the maltose binding protein domain
is linked to the amino-terminus of the polypeptide.
In certain embodiments, the bacterial host used for expression is
Escherichia coli.
In another embodiment, an enzymatic cleavage side is present
between the carboxyl-terminus of the maltose binding domain and the
amino-terminus of the polypeptides of the present disclosure. In
certain embodiments, said enzymatic cleavage side is a FXa cleavage
side.
In further embodiments, the polypeptides of the present are linked
to an antibody or an antibody fragment thereof. In certain
embodiments, said antibody fragment comprises a Fab fragment.
The antibody or antibody fragment thereof may have the same binding
specificity or a different binding specificity as the polypeptides
of the present disclosure.
The polypeptides of the present disclosure may be linked to the
antibody or an antibody fragment thereof, either by a direct
linkage to the amino- and/or carboxyl-terminus of the polypeptides
or by a spacer region comprising one or more amino acid residues at
the amino- and/or carboxyl-terminus of the polypeptides.
In certain embodiments, the polypeptides of the present disclosure
are linked to the carboxyl-terminus of the heavy chain of an
antibody. See FIG. 9A.
In other embodiments, the polypeptides of the present disclosure
are linked to the carboxyl-terminus of the light chain of an
antibody. See FIG. 9B.
In other embodiments, the polypeptides of the present disclosure
are linked to the carboxyl-terminus of a heavy chain antibody Fab
fragment. See FIG. 9C.
In other embodiments, the polypeptides of the present disclosure
are linked to the carboxyl-terminus of an antibody Fc-fragment
(CH2-CH3). See FIG. 9D.
In other embodiments, the polypeptides of the present disclosure
are linked to the amino-terminus of an antibody Fc-fragment
(CH2-CH3). See FIG. 9E.
In an embodiment, the present disclosure provides a
helix-turn-helix scaffold structure linked to an antibody or an
antibody fragment, wherein said helix-turn-helix scaffold structure
comprises a first and a second .alpha.-helical peptide that form an
anti-parallel, coiled-coil structure.
In an embodiment, the present disclosure provides a
helix-turn-helix scaffold structure linked to the carboxyl-terminus
of the heavy chains of an antibody, wherein said helix-turn-helix
scaffold structure comprises a first and a second .alpha.-helical
peptide that form an anti-parallel, coiled-coil structure.
In an embodiment, the present disclosure provides a
helix-turn-helix scaffold structure linked to the carboxyl-terminus
of the light chains of an antibody, wherein said helix-turn-helix
scaffold structure comprises a first and a second .alpha.-helical
peptide that form an anti-parallel, coiled-coil structure.
In an embodiment, the present disclosure provides a
helix-turn-helix scaffold structure linked to the carboxyl-terminus
of the heavy chain of an antibody Fab fragment, wherein said
helix-turn-helix scaffold structure comprises a first and a second
.alpha.-helical peptide that form an anti-parallel, coiled-coil
structure.
In an embodiment, the present disclosure provides a
helix-turn-helix scaffold structure linked to the amino-terminus of
an antibody Fc fragment, wherein said helix-turn-helix scaffold
structure comprises a first and a second .alpha.-helical peptide
that form an anti-parallel, coiled-coil structure.
In an embodiment, the present disclosure provides a
helix-turn-helix scaffold structure linked to the carboxyl-terminus
of an antibody Fc fragment, wherein said helix-turn-helix scaffold
structure comprises a first and a second .alpha.-helical peptide
that form an anti-parallel, coiled-coil structure.
Functionality
The polypeptides of the present disclosure may be used for the
prevention and treatment of diseases and disorders which are
mediated by biological pathway(s) in which the target molecule of
interest, against which the polypeptides are directed to, is
involved.
The polypeptides of the present disclosure can be used to prevent
or inhibit the interaction between one or more target molecules of
interest and their corresponding receptors or natural binding
partners, thereby preventing, inhibiting or reducing the signaling
pathways that are mediated by those target molecules of interest
and/or modulating the biological pathways and mechanisms in which
those target molecules of interest are involved.
Methods for assaying for functional activity may utilize binding
assays, such as the enzyme-linked immunosorbent assay (ELISA),
radioimmunoassay (RIA), fluorescence activated cell sorting (FACS)
and other methods that are well known in the art (see Hampton, R.
et al. (1990; Serological Methods a Laboratory Manual, APS Press,
St Paul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.
158:1211-1216). Alternatively, assays may test the ability of the
peptide mimetic in eliciting a biological response as a result of
binding to a biological target, either in vivo or in vitro. Such
assays include B cell and T cell proliferation assays, and
inhibition of proliferation assays (see Paul et al., (1991). Other
suitable assays will be known to those of skill in the art.
Pharmaceutical Compositions
In certain embodiments, the present disclosure provides
pharmaceutical compositions comprising one or more polypeptides
obtainable by the methods of the present disclosure and optionally
at least one pharmaceutically acceptable carrier together referred
to herein as pharmaceutical compositions. The pharmaceutical
compositions may further comprise at least one other
pharmaceutically active compound.
The pharmaceutical compositions of the present disclosure can be
used in the diagnosis, prevention and/or treatment of diseases and
disorders associated with a target molecule of interest.
In particular, the present disclosure provides pharmaceutical
compositions comprising polypeptides according to the present
disclosure that are suitable for prophylactic, therapeutic and/or
diagnostic use in a warm-blooded animal, and in particular in a
mammal, and more in particular in a human being.
Generally, the polypeptides of the present disclosure may be
formulated as a pharmaceutical preparation or compositions
comprising at least one polypeptide according to the present
disclosure and at least one pharmaceutically acceptable carrier,
diluent or excipient and/or adjuvant, and optionally one or more
further pharmaceutically active polypeptides and/or compounds. Such
a formulation may be suitable for oral, parenteral, topical
administration or for administration by inhalation.
In particular, the polypeptides of the present disclosure may be
used in combination with other pharmaceutically active compounds
that are or can be used for the prevention and/or treatment of the
diseases and disorders in which a target molecule of interest is
involved, as a result of which a synergistic effect may or may not
be obtained. Examples of such compounds, as well as routes, methods
and pharmaceutical formulations or compositions for administering
them will be clear to the clinician.
In an embodiment, the present disclosure provides a pharmaceutical
composition comprising one or more polypeptides of the present
disclosure for use in the prevention and/or treatment of a disorder
or condition associated with the undesired presence of a target
molecule of interest specifically bound by the one or more
polypeptides
In an embodiment, the present disclosure provides a pharmaceutical
composition comprising one or more polypeptides of the present
disclosure for the use as a medicament.
In an embodiment, the disclosure provides a pharmaceutical
composition comprising one or more polypeptides of the present
disclosure for use in the prevention and/or treatment of autoimmune
diseases, inflammatory diseases, cancer, neovascular diseases,
infectious diseases, thrombosis, myocardial infarction, and/or
diabetes.
In an further embodiment, the disclosure provides a method for the
treatment of autoimmune diseases, inflammatory diseases, cancer,
neovascular diseases, infectious diseases, thrombosis, myocardial
infarction, and/or diabetes in a subject in need thereof using a
pharmaceutical composition comprising one or more polypeptides of
the present disclosure.
EXAMPLES
Example 1: Design of the Library
The design of the library is based on a combined approach taking
into account statistical, structural and rational factors.
This includes the analysis of the most abundant amino acid residues
found in natural .alpha.-helical structures. Such amino acids may
have favorable biophysical properties that would lead to more
efficient development and increase the safety and efficacy of the
resulting polypeptides in patients. The abundancy of natural
occurring amino acids in .alpha.-helices was obtained from
literature, such as Aurora et al. (Protein Science (1998), 7:21-38)
and Pace et al. (Biophysical Journal (1998), Vol. 75, 422-427).
These data were used to catalog residue preferences at the amino-
and carboxyl-terminal ends of .alpha.-helices, as well as to
catalog residue preferences found more in the central region of
.alpha.-helices. A particular focus was on those amino acids that
occur at positions that are relevant for the formation of the
.alpha.-helical coiled-coil structure in aqueous solution.
Data was subsequently transferred into a template to design
potential .alpha.-helical reference sequences. Such a reference
sequence has a length of 15 amino acid residues and comprises two
consecutive heptad sequences. For each of said 15 amino acid
residue positions, the five to six most frequently occurring amino
acid residues were considered as a basis for designing two
independent .alpha.-helical peptide sequences being able to from a
stabilized anti-parallel orientated coiled-coil structure in
aqueous solution.
In order to select the optimal reference sequence for the
generation of a polypeptide library according of the present
disclosure, additional structural consideration for promoting helix
formation and stabilizing the .alpha.-helical coiled-coil structure
were taken into account to select the most appropriate amino acid
residue at each position.
Accordingly, a reference polypeptide sequence (HTHdes2) comprising
two .alpha.-helical peptides and a linker segment as depicted in
FIG. 3, was designed and selected for synthesis and testing for
.alpha.-helical content by circular dichroism (CD) spectra
measurements. The obtained data confirmed that the HTHdes2
reference sequence resulted in a high degree of .alpha.-helical
structure and a low content of random coiled structures in
solution. Additional studies revealed that the HTHdes2 sequence was
extremely resistant towards thermal and chemical denaturation. See
also Example 12.
In addition, an in silico T cell epitope screening for the HTHdes2
reference sequence (Lonza, The Epibase.TM. In Silico tool) revealed
a low to no immunogenicity risk for the generated construct.
The HTHdes2 reference sequence was subsequently used for the
construction of the polypeptide library according to the present
disclosure.
In a first step, the inventors had to decide which specific amino
acid positions on the HTHdes2 reference sequence should to be used
for diversification without destabilizing the .alpha.-helical
secondary structure of the two HTH forming peptides and without
compromising the formation of the coiled-coil structure.
Stabilizing a HTH structure may be achieved by diversifying only
one of the two alpha helical peptides as it has been described by
Fujii et al (Biochemistry, 47, 6745-6751 (2008)). In this scenario,
the second non-diversified peptide retains its .alpha.-helical
structure and stabilizes the helix-turn-helix structure. However,
diversification of only one peptide significantly limits the
achievable library size and narrows down the interaction interphase
between the polypeptides of the library and their bound target
molecules of interest.
Thus, the inventors decided to diversify both .alpha.-helical
peptides. The variable positions present on both .alpha.-helical
peptides contribute to an enlarged interaction interphase between a
particular polypeptide and its target antigen, thus resulting in
improved specificity and affinity.
As described herein and as shown in FIGS. 1 and 3, the amino acid
positions, which were primarily selected for diversification, were
heptad positions `b` and `e` of Helix-1 and Helix-2. Since each
helix comprises two heptads, 4 amino acid position are diversified
per helix. These amino acid positions, herein referred to as Var1
are displayed in the same relative parallel orientation and are the
key solvent exposed residues for interacting with a target antigen
of interest (FIGS. 2A and 2B). Because of this predominant role,
the respective positions were fully diversified with 17 naturally
occurring amino acids, leaving out only G, P and C which are known
to break .alpha.-helical structures.
The assembly of the two .alpha.-helical peptides provides for a
total of eight diversified amino acid residues, all of which are
displayed in the same relative parallel orientation out of the HTH
scaffold structure with a slight spatial off-set of the diversified
positions within the two aligned peptides. Consequently, a wide and
flat interaction interface is formed over the whole length of the
HTH scaffold structure, which allows an optimal protein-protein
interaction with a target antigen of interest.
In order to further increase the library size and the interaction
interface, additional amino acid residues within the HTHdes2
reference sequence were considered for diversification. Amino acid
residues present at heptad positions f are also orientated towards
the interaction interface even though not completely in a parallel
fashion. Based on the local geometry, the orientation of residues
at heptad positions f appear slightly angulated (FIG. 1 and FIGS.
2A and 2B) but the residues may still be able to interact with a
target antigen. Thus, 4 of said heptad f positions were selected
for further diversification and were named Var2 and Var3.
Based on the angulated orientation of the residues towards the
interaction interface, amino acids with charged or polar side
chains like E, D, K, R, N, Q, and H were considered as the optimal
residues for diversification of Var2 and Var3. In this scenario,
the terminally located polar or charged functional groups of each
residue is capable of interacting with the surrounding solvent
whereas the nonpolar carbon backbone can interact with the target
antigen. Final selection of the 3 amino acid residues used for
diversification of Var2 and Var3 was driven by their natural
occurrence in .alpha.-helices at corresponding positions as
described by Aurora et al. (Protein Science (1998), 7:21-38) and
Pace et al. (Biophysical Journal (1998), Vol. 75, 422-427).
Therefore, a slightly different amino acid residue mixture was used
for Var2 and Var3. The theoretical library size therefore amounts
to 5, 6.times.10.sup.11 library members.
In summary, the library of the present disclosure was built to
maximize the number of diversified positions without compromising
the stabilizing .alpha.-helical structures which form the HTH
scaffold structure of the present disclosure.
Example 2: Generation of the HTH-lib1 Library
The DNA fragments containing the polypeptide library sequence were
synthesized as follows: The flanking constant regions comprising a
signal sequence, epitope tag and spacer regions were synthesized by
gene synthesis. The polypeptide library encoding the polypeptide
sequence with 12 diversified amino acid positions was synthesized
with the Slonomics technology. The resulting 279 bp synthetic
linear DNA fragments comprising the polypeptide library and the
flanking constant regions were cloned into the pPEPdisC3fl_HTH-lib1
display vector (as described in WO2015166036 with minor
modifications).
Example 3: Determination of Display Rate
Display of polypeptides on the produced phage was evaluated by
Western Blot. After SDS-PAGE separation, proteins were detected
using an antibody against the FLAG epitope (M2, Sigma-Aldrich) as
this epitope tag is encoded by the pIII (fl)-polypeptide library
and therefore part of the displayed peptides. For detection of
total pIII, an anti-pIII antibody (MiBiTec) was used. Helper phages
displayed approx. 2-3 library peptides per phage and on hyper
phages all 5 pIII copies carry the library peptides.
Furthermore, display of polypeptides on the produced phage was
qualitatively evaluated by ELISA, utilizing an anti-M13 antibody
(GE Healthcare) for phage capturing and two antibodies for specific
detection. A monoclonal anti-M13 conjugated to HRP (Amersham) and a
monoclonal antibody against the FLAG epitope conjugated to AP
(Sigma).
The results confirmed a high display rate of library
polypeptides.
Example 4: Quality Control
Another important aspect is the evaluation of the quality and
functionality of the polypeptide HTH-lib1 library. A qualitative
assessment of the phage library, with respect to amino acid
distribution, frequency and redundancy was carried out using Sanger
sequencing.
175 clones were analyzed from the library design shown in FIG. 3
using Sanger sequencing. Sequencing results are shown in FIG. 4A.
FIG. 4A shows the position and distribution of the indicated amino
acids at diversified positions in Helix-1 and Helix-2,
respectively.
Of the 175 individual clones sampled, a well-balanced distribution
of the natural occurring amino acids except G, P and C was
identified for Var1. Similarly, a well-balanced distribution of the
amino acids R, Q and E for Var2 and of amino acids R, Q and H for
Var3 could be confirmed.
FIG. 4B shows the expected amino acid distribution in comparison to
the sequencing results (given in percentage). These results
demonstrate that the composition of the synthesized library is
essentially identical to the library design.
Example 5: Pannings and Screenings
The suitability of the polypeptide library HTH-lib1 disclosed
herein for the identification of potential therapeutic peptides was
analyzed using available model antigens.
The HTH-lib1 library was used for test selections against model
Target-X. Target-X was used as a hlgFc fusion protein. Selections
were performed in solution using Protein G coupled magnetic beads
(Dynabeads Protein G, Life Technologies) which are able to capture
the antigen-phage complex by the human Fc-tag of the antigen.
The polypeptide library was handled according to published standard
protocols for phage display based peptide selections (Zwick, M. B.,
Menendez, A., Bonnycastle, L. L. C. and Scott, J. K. (2001). In C.
F. Barbas, D. R. Burton, J. K. Scott and G. J. Silverman, (Eds.),
Phage Display: A Laboratory Manual (pp. 18.1-18.44). New York: Cold
Spring Harbor Laboratory Press) with minor adjustments in terms of
selection stringency and adaptation to phagemide vector system.
The test selections were performed over 3 subsequent enrichment
rounds with monitoring of specific sequences by conventional
sequencing. In short, all pannings were completed with various
antigen concentrations (100 nM for round 1, 50 nM for round 2, and
25 nM for round 3) under standard washing conditions. The target
protein was incubated with pre-adsorbed phages. Washing of the
coated magnetic Dynabeads was carried out with a magnetic particle
separator and incubations were done by overhead rotation in low
binding tubes. Subsequently, the specifically bound phages were
eluted using Glycine/HCl.
E. coli TG1F' with an OD600 nm of 0.6-0.8 was added to the phage
eluates of each selection and was incubated in an incubator without
shaking. After infection bacteria were plated out evenly on two
large LB/Chloramphenicol/Glucose agar plates for each selection and
incubated overnight at 37.degree. C. and Glycerol phage stocks were
prepared.
For the following panning rounds bacterial suspensions of each pool
were collected and used to propagate phages for an additional
panning round as described above.
After each round of panning the phage titer was determined. The
expected range goes from 1.0E+10-1.0E+12 phage/ml for the input and
1.0E+07-1.0E+09 phage/ml for the output. Table 3 shows the output
after each round of panning and all values are in the expected
range.
TABLE-US-00004 TABLE 3 Phage output titers Phage Phage Phage
Panning Output Output Output 3rd Library Strategy Target 1st round
2nd round round HTH-lib1 Solution Target-X/hFc 1.11E+07 1.45E+07
4.06E+08
After completion of the panning rounds phage output pools were
subcloned via PCR into an expression vector to facilitate the
cytoplasmic expression of the polypeptides in E. coli. Expression
of single clones resulted in the production of polypeptides that
were N-terminally linked to the Maltose Binding Protein (MBP) and
that include a protease cleavage site.
To check for target specificity, an ELISA screen was carried out by
capturing the Fc-tagged cynomolgus and human Target-X protein as
well as an unrelated Fc-tagged protein on the surface of a
microtiter plate pre-coated with an anti-human Fc specific antibody
(Jackson Immuno Research) and E. coli lysates containing the
polypeptides obtained from the panning outputs. Bound polypeptides
were detected by the encoded FLAG tag using anti-FLAG detection
(anti-FLAG-AP, Sigma Aldrich). To analyze MBP-polypeptide fusion
expression, anti-His capture (R&D Systems) and anti-MBP
detection (Abcam) was applied.
In total, 280 polypeptides were identified recognizing the
cynomolgus Target-X/Fc fusion protein in ELISA. Furthermore, almost
all cynomolgus positive clones (98%) revealed cross-reactivity to
the human Target-X protein but no binding to an irrelevant
Fc-fusion protein.
Sequence analysis and ELISA of panning outputs from subsequent
panning rounds revealed an enrichment of specific binders, i.e.
binders that bind to the target protein but not to unrelated
proteins.
Example 6: Sequencing Results of Target-X Specific Polypeptides
Sanger sequencing was performed to analyze the binders that are
specific for the human and the cynomolgus Target-X/Fc fusion
protein (see Example 5). The sequencing results revealed a diverse
number of target specific polypeptides as depicted in FIG. 9. This
demonstrates that the library of the present disclosure can be used
to identify a large variety of polypeptides that are specific for
the target protein of interest.
As a next step, selected polypeptides from the screening campaign
were produced in larger scale in order to characterize them in more
detail for properties, such as ELISA and cell binding, affinity,
and functional activity in a relevant in vitro assay.
Example 7: Characterization of Purified Target-X Specific
Polypeptides for ELISA Binding
Binding to Human and Cynomolgus Target-X was Tested in an
ELISA.
Methods:
1.5 .mu.g/ml of each Fc fusion protein was captured via an
anti-human-Fc-capture (Jackson Immuno Research) specific antibody
on Maxisorp plates and bound MBP-polypeptide fusions were detected
using an anti-FLAG detection antibody.
Results:
All 18 polypeptides showed significant and specific binding to
both, recombinant cynomolgus and human Target-X/Fc proteins.
EC.sub.50 values range from the single to triple digit nanomolar
range.
TABLE-US-00005 TABLE 4 ELISA binding to different Target-X species.
ELISA/EC.sub.50 (nM) cyno human HTH00024 12.6 478 HTH00025 633 459
HTH00029 96 98 HTH00031 80 86 HTH00032 203 337 HTH00033 738 678
HTH00034 3.1 3.1 HTH00035 28 20 HTH00036 85 79 HTH00037 65 109
HTH00039 54 80 HTH00040 90 150 HTH00041 111 114 HTH00042 19.4 15.7
HTH00043 154 194 HTH00044 485 429 HTH00055 93 94 HTH00056 196
503
These results confirms the highly specific nature of the
polypeptides isolated from the library of the present
disclosure.
Example 8: Characterization of Target-X Specific Polypeptides for
Cell Binding (FACS)
Cell Binding to Cynomolgus Target-X Expressed on CHO Cells was
Analyzed by FACS.
Methods:
CHO cells stable transfected with cynomolgus Target-X were adjusted
to 2.times.10.sup.6 cells/ml in PBS/3% FCS/0.02% NaN.sub.3 (FACS
buffer). FACS staining was performed in V-bottom 96-well microtiter
and 1.times.10.sup.5 cells per well were mixed with purified
polypeptides, diluted in FACS buffer and incubated on ice for 1 h.
Cells were then washed 4.times. with 150 .mu.l FACS buffer/well and
taken up in 50 .mu.l rabbit anti-MBP (Abcam), diluted 1:10000 in
FACS buffer. After 1 h incubation on ice cells were washed 4.times.
with FACS buffer and taken up in 50 .mu.l phycoerythrin-conjugated
anti-rabbit secondary antibody (Jackson Immuno Research), diluted
1:100 in FACS buffer. After 30 min incubation on ice, cells were
washed 4.times. with FACS buffer, resuspended in 100 .mu.l FACS
buffer and cell surface binding of cynomolgus Target-X specific
antibodies was measured via FL2 fluorescence intensity of cells in
FACSArray (Becton Dickinson).
Results:
19 purified polypeptides showed specific cell binding to cynomolgus
Target-X expressed on CHO cells with EC.sub.50 values ranging from
the single to triple digit nanomolar range.
TABLE-US-00006 TABLE 5 Cell binding (FACS) to cynomolgus
Target-X-CHO cells. Cell Binding/EC.sub.50 (nM) HTH00024 17.5
HTH00025 253 HTH00027 235 HTH00029 333 HTH00031 146 HTH00032 26
HTH00033 412 HTH00034 4.9 HTH00035 688 HTH00036 144 HTH00037 247
HTH00039 93 HTH00040 14.6 HTH00041 32 HTH00042 82 HTH00043 159
HTH00044 140 HTH00055 580 HTH00056 53
Again, this result confirms the highly specific nature of the
polypeptides isolated from the library of the present disclosure.
Polypeptides are also able to bind to the target protein on whole
cells.
Example 9: Affinity Determination of a Target-X Specific
Polypeptide Using Surface Plasmon Resonance
Kinetic characterization of the interaction between cynomolgus
Target-X/Fc and the Target-X-specific polypeptide was carried out
in ligand-capture format, with the polypeptide being applied as
analyte in solution.
Methods:
A Biacore CM5 sensor chip (GE Healthcare) was covalently modified
to generate a high-density capture surface specific for human Fc.
All flowcells were immobilized with approx. 3500 RU MabSelect
SuRe.TM. Ligand (GE Healthcare; 50 .mu.g/mL in 10 mM Acetate buffer
pH 4.5) using standard EDC-NHS amine coupling chemistry. 10 mM
HBS-EP+ pH7.4 (GE Healthcare) was used as running buffer and sample
diluent. During kinetic characterization, Target-X/Fc was captured
onto an anti-human-Fc specific flowcell (20 nM; 75 s injection;
capture level approx. 250 RU), followed by analyte injection
(association) for 180 s and dissociation (variable times; up to 600
s; flow rate 40 .mu.L/min). A 2-fold serial dilution series of
analyte concentrations from 1.37 to 1000 nM was analyzed. At the
end of each cycle, captured ligand and bound analyte were removed
with 10 mM Glycine/HCl pH 1.5 by 2 injections a 30 s. Blank
injections (analyte concentration=0 nM) were included, and
subtracted for double referencing. The resulting sensorgrams were
evaluated with Biacore T200 Evaluation Software 3.0 (GE Healthcare)
using 1:1 kinetic and steady state models.
Results:
The affinity of the Target-X specific polypeptide was measured on
captured cynomolgus Target-X/Fc in a Biacore system. The affinity
of the polypeptide was 36 nM in a kinetic model and 45 nM in a
steady state model (Table 6).
This demonstrates that the polypeptides of the present disclosure
are not just highly specific but also bind with a high affinity to
their targets.
Example 10: Cynomolgus Target-X/Receptor Binding Inhibition Assay
(ELISA)
Methods:
10 .mu.g/ml of the Target-X related recombinant receptor protein
was coated on a MSD-plate and blocked with milk powder. Different
concentrations of a Target-X specific MBP-free polypeptide were
mixed with 0.5 .mu.g/ml cynomolgus Target-X/Fc and incubated for 30
min at RT. After washing the blocked MSD-plate, the
polypeptide-Target-X/Fc mixtures were applied to the plate and
incubated for 1 h at RT. After washing, receptor bound Target-X was
detected using an ECL-conjugated anti-human Fc specific antibody
(1:2000, Jackson Immuno Research). Inhibition of the specific
receptor/target interaction by the polypeptide lead to decreasing
signals for Target-X/Fc bound to its receptor.
Results:
A purified cynomolgus Target-X specific MBP-free polypeptide was
analyzed for its potential inhibitory in vitro activity on
receptor/cynomolgus Target-X interaction. The polypeptide showed
significant receptor/cynomolgus Target-X interaction inhibition in
an ELISA with an IC.sub.50 value of 14.7 nM.
TABLE-US-00007 TABLE 6 Summary of the results from the in vitro
characterization MBP-free peptide: ELISA (EC50) 6.3 nM FACS (EC50)
6.5 nM SPR (Kd) 45 nM Inhibition (IC50) 14.7 nM
Example 11: Structural Analysis of the Helix-Turn-Helix Design of
Target-X Specific Polypeptides
.alpha.-helical content of a Target-X specific polypeptide was
analyzed by measuring circular dichroism (CD) spectra in 10 mM
phosphate buffer (pH 7.2) with a peptide concentration of 0.1
mg/ml, using a Chirascan Plus CD-Spectrophotometer (Applied
Photophysics).
As shown in FIG. 8, the polypeptide exhibited double minima at 208
and 222 nm, indicating that the polypeptide has high content of
.alpha.-helical structure in aqueous solution.
In addition, the thermal stability was determined by incubating the
polypeptide in aqueous solution at increasing temperatures ranging
from 20.degree. C. to 90.degree. C. Analysis of the CD spectra
revealed that the polypeptide showed almost no structural unfolding
even after treatment at 90.degree. C. (FIG. 8).
Example 12: Stability of the Helix-Turn-Helix Design
Stability against chemical denaturation was determined by
incubating a polypeptide comprising the HTHdes2 reference sequence,
in 2 and 4 molar Guanidin-Hydrochlorid (GdmHCL) at 20.degree. C.
Even treatment with 4M GdmHCL did not resulted in complete
unfolding of the polypeptide. In a further experiment, the
polypeptide was treated with 2M GdmHCL plus increasing temperatures
ranging from 22.degree. C. to 90.degree. C. Again, even after
treatment with 2M GdmHCL at 90.degree. C., no complete unfolding of
the polypeptide could be observed.
Stability against low pH denaturation was examined for a sequence
variant of the HTHdes2 reference sequence polypeptide, wherein 6
amino acid residues in Helix-2 were exchanged. Treatment of the
polypeptide in pH 2 for 60 min. with or without subsequent
neutralization to a pH 7.2 did not altered the .alpha.-helical
content of the polypeptide.
All the performed studies clearly demonstrate the superior
stability properties of the polypeptides, which can be isolated
from the library of the present disclosure.
SEQUENCE LISTINGS
1
67115PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide"VARIANT(1)..(1)/replace="T" or "N" or
"S" or "P"VARIANT(2)..(2)/replace="P" or "Q" or "W" or
"D"MOD_RES(3)..(3)Any amino acid residue having a side chain
exhibiting a hydrophobicity of greater than
0.62VARIANT(4)..(4)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"VARIANT(5)..(5)/replace="A" or "I" or "Q" or
"R"MOD_RES(6)..(6)Any amino acid residue having a side chain
exhibiting a hydrophobicity of greater than
0.62VARIANT(7)..(7)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"VARIANT(8)..(8)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"VARIANT(9)..(9)/replace="L" or "R" or "M" or "K" or
"E"MOD_RES(10)..(10)Any amino acid residue having a side chain
exhibiting a hydrophobicity of greater than
0.62VARIANT(11)..(11)/replace="D" or "E" or "F" or "H" or "I" or
"K" or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W"
or "Y"VARIANT(12)..(12)/replace="L" or "A" or "W" or "F" or
"K"MOD_RES(13)..(13)Any amino acid residue having a side chain
exhibiting a hydrophobicity of greater than
0.62VARIANT(14)..(14)/replace="D" or "E" or "F" or "H" or "I" or
"K" or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W"
or "Y"VARIANT(15)..(15)/replace="D" or "E" or "F" or "H" or "I" or
"K" or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W"
or "Y"MISC_FEATURE(1)..(15)/note="Variant residues given in the
sequence have no preference with respect to those in the
annotations for variant positions" 1Asp Glu Xaa Ala Met Xaa Ala Ala
Ala Xaa Ala Met Xaa Ala Ala1 5 10 15260PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide"VARIANT(1)..(1)/replace="T" or "N" or "S" or
"P"VARIANT(2)..(2)/replace="P" or "Q" or "W" or
"D"MOD_RES(3)..(3)Any amino acid residue having a side chain
exhibiting a hydrophobicity of greater than
0.62VARIANT(4)..(4)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"VARIANT(5)..(5)/replace="A" or "I" or "Q" or
"R"MOD_RES(6)..(6)Any amino acid residue having a side chain
exhibiting a hydrophobicity of greater than
0.62VARIANT(7)..(7)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"VARIANT(8)..(8)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"VARIANT(9)..(9)/replace="L" or "R" or "M" or "K" or
"E"MOD_RES(10)..(10)Any amino acid residue having a side chain
exhibiting a hydrophobicity of greater than
0.62VARIANT(11)..(11)/replace="D" or "E" or "F" or "H" or "I" or
"K" or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W"
or "Y"VARIANT(12)..(12)/replace="L" or "A" or "W" or "F" or
"K"MOD_RES(13)..(13)Any amino acid residue having a side chain
exhibiting a hydrophobicity of greater than
0.62VARIANT(14)..(14)/replace="D" or "E" or "F" or "H" or "I" or
"K" or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W"
or "Y"VARIANT(15)..(15)/replace="D" or "E" or "F" or "H" or "I" or
"K" or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W"
or "Y"MOD_RES(16)..(45)Any amino acid or not
presentMISC_FEATURE(16)..(45)/note="This region may encompass 1 to
30 amino acid residues"VARIANT(46)..(46)/replace="T" or "N" or "S"
or "P"VARIANT(47)..(47)/replace="P" or "Q" or "W" or
"D"MOD_RES(48)..(48)Any amino acid residue having a side chain
exhibiting a hydrophobicity of greater than
0.62VARIANT(49)..(49)/replace="D" or "E" or "F" or "H" or "I" or
"K" or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W"
or "Y"VARIANT(50)..(50)/replace="A" or "I" or "Q" or
"R"MOD_RES(51)..(51)Any amino acid residue having a side chain
exhibiting a hydrophobicity of greater than
0.62VARIANT(52)..(52)/replace="D" or "E" or "F" or "H" or "I" or
"K" or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W"
or "Y"VARIANT(53)..(53)/replace="D" or "E" or "F" or "H" or "I" or
"K" or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W"
or "Y"VARIANT(54)..(54)/replace="L" or "R" or "M" or "K" or
"E"MOD_RES(55)..(55)Any amino acid residue having a side chain
exhibiting a hydrophobicity of greater than
0.62VARIANT(56)..(56)/replace="D" or "E" or "F" or "H" or "I" or
"K" or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W"
or "Y"VARIANT(57)..(57)/replace="L" or "A" or "W" or "F" or
"K"MOD_RES(58)..(58)Any amino acid residue having a side chain
exhibiting a hydrophobicity of greater than
0.62VARIANT(59)..(59)/replace="D" or "E" or "F" or "H" or "I" or
"K" or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W"
or "Y"VARIANT(60)..(60)/replace="D" or "E" or "F" or "H" or "I" or
"K" or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W"
or "Y"MISC_FEATURE(1)..(60)/note="Variant residues given in the
sequence have no preference with respect to those in the
annotations for variant positions" 2Asp Glu Xaa Ala Met Xaa Ala Ala
Ala Xaa Ala Met Xaa Ala Ala Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Glu Xaa 35 40 45Ala Met Xaa Ala Ala
Ala Xaa Ala Met Xaa Ala Ala 50 55 60360PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide"MOD_RES(3)..(3)Any amino acid residue having a side
chain exhibiting a hydrophobicity of greater than
0.62VARIANT(4)..(4)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"MOD_RES(6)..(6)Any amino acid residue having a side chain
exhibiting a hydrophobicity of greater than
0.62VARIANT(7)..(7)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"VARIANT(8)..(8)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"MOD_RES(10)..(10)Any amino acid residue having a side chain
exhibiting a hydrophobicity of greater than
0.62VARIANT(11)..(11)/replace="D" or "E" or "F" or "H" or "I" or
"K" or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W"
or "Y"MOD_RES(13)..(13)Any amino acid residue having a side chain
exhibiting a hydrophobicity of greater than
0.62VARIANT(14)..(14)/replace="D" or "E" or "F" or "H" or "I" or
"K" or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W"
or "Y"VARIANT(15)..(15)/replace="D" or "E" or "F" or "H" or "I" or
"K" or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W"
or "Y"MOD_RES(16)..(45)Any amino acid or not
presentMISC_FEATURE(16)..(45)/note="This region may encompass 1 to
30 amino acid residues"MOD_RES(48)..(48)Any amino acid residue
having a side chain exhibiting a hydrophobicity of greater than
0.62VARIANT(49)..(49)/replace="D" or "E" or "F" or "H" or "I" or
"K" or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W"
or "Y"MOD_RES(51)..(51)Any amino acid residue having a side chain
exhibiting a hydrophobicity of greater than
0.62VARIANT(52)..(52)/replace="D" or "E" or "F" or "H" or "I" or
"K" or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W"
or "Y"VARIANT(53)..(53)/replace="D" or "E" or "F" or "H" or "I" or
"K" or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W"
or "Y"MOD_RES(55)..(55)Any amino acid residue having a side chain
exhibiting a hydrophobicity of greater than
0.62VARIANT(56)..(56)/replace="D" or "E" or "F" or "H" or "I" or
"K" or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W"
or "Y"MOD_RES(58)..(58)Any amino acid residue having a side chain
exhibiting a hydrophobicity of greater than
0.62VARIANT(59)..(59)/replace="D" or "E" or "F" or "H" or "I" or
"K" or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W"
or "Y"VARIANT(60)..(60)/replace="D" or "E" or "F" or "H" or "I" or
"K" or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W"
or "Y"MISC_FEATURE(1)..(60)/note="Variant residues given in the
sequence have no preference with respect to those in the
annotations for variant positions" 3Asp Glu Xaa Ala Gln Xaa Ala Ala
Glu Xaa Ala Lys Xaa Ala Ala Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Glu Xaa 35 40 45Ala Ala Xaa Ala Ala
Lys Xaa Ala Met Xaa Ala Ala 50 55 60460PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide"VARIANT(1)..(1)/replace="T" or "N" or "S" or
"P"VARIANT(2)..(2)/replace="P" or "Q" or "W" or
"D"VARIANT(3)..(3)/replace="V" or "I"VARIANT(4)..(4)/replace="D" or
"E" or "F" or "H" or "I" or "K" or "L" or "M" or "N" or "Q" or "R"
or "S" or "T" or "V" or "W" or "Y"VARIANT(5)..(5)/replace="A" or
"I" or "Q" or "R"VARIANT(6)..(6)/replace="V" or
"I"VARIANT(7)..(7)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"VARIANT(8)..(8)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"VARIANT(9)..(9)/replace="L" or "R" or "M" or "K" or
"E"VARIANT(10)..(10)/replace="V" or
"I"VARIANT(11)..(11)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"VARIANT(12)..(12)/replace="L" or "A" or "W" or "F" or
"K"VARIANT(13)..(13)/replace="V" or
"I"VARIANT(14)..(14)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"VARIANT(15)..(15)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"MOD_RES(16)..(45)Any amino acid or not
presentMISC_FEATURE(16)..(45)/note="This region may encompass 1 to
30 amino acid residues"VARIANT(46)..(46)/replace="T" or "N" or "S"
or "P"VARIANT(47)..(47)/replace="P" or "Q" or "W" or
"D"VARIANT(48)..(48)/replace="V" or
"I"VARIANT(49)..(49)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"VARIANT(50)..(50)/replace="A" or "I" or "Q" or
"R"VARIANT(51)..(51)/replace="V" or
"I"VARIANT(52)..(52)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"VARIANT(53)..(53)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"VARIANT(54)..(54)/replace="L" or "R" or "M" or "K" or
"E"VARIANT(55)..(55)/replace="V" or
"I"VARIANT(56)..(56)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"VARIANT(57)..(57)/replace="L" or "A" or "W" or "F" or
"K"VARIANT(58)..(58)/replace="V" or
"I"VARIANT(59)..(59)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"VARIANT(60)..(60)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"MISC_FEATURE(1)..(60)/note="Variant residues given in the
sequence have no preference with respect to those in the
annotations for variant positions" 4Asp Glu Leu Ala Met Leu Ala Ala
Ala Leu Ala Met Leu Ala Ala Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Glu Leu 35 40 45Ala Met Leu Ala Ala
Ala Leu Ala Met Leu Ala Ala 50 55 60560PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide"VARIANT(1)..(1)/replace="T" or "N" or "S" or
"P"VARIANT(2)..(2)/replace="P" or "Q" or "W" or
"D"MOD_RES(3)..(3)Any amino acid residue having a side chain
exhibiting a hydrophobicity of greater than
0.62VARIANT(4)..(4)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"VARIANT(5)..(5)/replace="A" or "I" or "Q" or
"R"MOD_RES(6)..(6)Any amino acid residue having a side chain
exhibiting a hydrophobicity of greater than
0.62VARIANT(7)..(7)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"VARIANT(8)..(8)/replace="R" or "Q"VARIANT(9)..(9)/replace="L" or
"R" or "M" or "K" or "E"MOD_RES(10)..(10)Any amino acid residue
having a side chain exhibiting a hydrophobicity of greater than
0.62VARIANT(11)..(11)/replace="D" or "E" or "F" or "H" or "I" or
"K" or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W"
or "Y"VARIANT(12)..(12)/replace="L" or "A" or "W" or "F" or
"K"MOD_RES(13)..(13)Any amino acid residue having a side chain
exhibiting a hydrophobicity of greater than
0.62VARIANT(14)..(14)/replace="D" or "E" or "F" or "H" or "I" or
"K" or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W"
or "Y"VARIANT(15)..(15)/replace="Q" or "H"MOD_RES(16)..(45)Any
amino acid or not presentMISC_FEATURE(16)..(45)/note="This region
may encompass 1 to 30 amino acid
residues"VARIANT(46)..(46)/replace="T" or "N" or "S" or
"P"VARIANT(47)..(47)/replace="P" or "Q" or "W" or
"D"MOD_RES(48)..(48)Any amino acid residue having a side chain
exhibiting a hydrophobicity of greater than
0.62VARIANT(49)..(49)/replace="D" or "E" or "F" or "H" or "I" or
"K" or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W"
or "Y"VARIANT(50)..(50)/replace="A" or "I" or "Q" or
"R"MOD_RES(51)..(51)Any amino acid residue having a side chain
exhibiting a hydrophobicity of greater than
0.62VARIANT(52)..(52)/replace="D" or "E" or "F" or "H" or "I" or
"K" or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W"
or "Y"VARIANT(53)..(53)/replace="R" or
"Q"VARIANT(54)..(54)/replace="L" or "R" or "M" or "K" or
"E"MOD_RES(55)..(55)Any amino acid residue having a side chain
exhibiting a hydrophobicity of greater than
0.62VARIANT(56)..(56)/replace="D" or "E" or "F" or "H" or "I" or
"K" or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W"
or "Y"VARIANT(57)..(57)/replace="L" or "A" or "W" or "F" or
"K"MOD_RES(58)..(58)Any amino acid residue having a side chain
exhibiting a hydrophobicity of greater than
0.62VARIANT(59)..(59)/replace="D" or "E" or "F" or "H" or "I" or
"K" or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W"
or "Y"VARIANT(60)..(60)/replace="Q" or
"H"MISC_FEATURE(1)..(60)/note="Variant residues given in the
sequence have no preference with respect to those in the
annotations for variant positions" 5Asp Glu Xaa Ala Met Xaa Ala Glu
Ala Xaa Ala Met Xaa Ala Arg Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Glu Xaa 35 40 45Ala Met Xaa Ala Glu
Ala Xaa Ala Met Xaa Ala Arg 50 55 6065PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 6Gln Gly Val Asp Ser1 5735PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide"VARIANT(4)..(4)/replace="D" or "E" or "F" or "H" or "I"
or "K" or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or
"W" or "Y"VARIANT(7)..(7)/replace="D" or "E" or "F" or "H" or "I"
or "K" or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or
"W" or "Y"VARIANT(8)..(8)/replace="D" or "E" or "F" or "H" or "I"
or "K" or
"L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"VARIANT(11)..(11)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"VARIANT(14)..(14)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"VARIANT(15)..(15)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"VARIANT(24)..(24)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"VARIANT(27)..(27)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"VARIANT(28)..(28)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"VARIANT(31)..(31)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"VARIANT(34)..(34)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"VARIANT(35)..(35)/replace="D" or "E" or "F" or "H" or "I" or "K"
or "L" or "M" or "N" or "Q" or "R" or "S" or "T" or "V" or "W" or
"Y"MISC_FEATURE(1)..(35)/note="Variant residues given in the
sequence have no preference with respect to those in the
annotations for variant positions" 7Asp Glu Leu Ala Gln Leu Ala Ala
Glu Leu Ala Lys Leu Ala Ala Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Ala Ala Leu Ala Ala Lys Leu Ala Met 20 25 30Leu Ala Ala
35835PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 8Asp Glu Leu Ala Gln Leu Glu Arg
Glu Leu Met Lys Leu Lys Ala Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Glu Ala Leu Ala Arg Lys Leu Ala Met 20 25 30Leu Ala Arg
35935PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide"VARIANT(4)..(4)/replace="R" or "N"
or "D" or "E" or "Q" or "H" or "I" or "L" or "K" or "M" or "F" or
"S" or "T" or "W" or "Y" or "V"VARIANT(7)..(7)/replace="R" or "N"
or "D" or "E" or "Q" or "H" or "I" or "L" or "K" or "M" or "F" or
"S" or "T" or "W" or "Y" or "V"VARIANT(8)..(8)/replace="Q" or
"E"VARIANT(11)..(11)/replace="R" or "N" or "D" or "E" or "Q" or "H"
or "I" or "L" or "K" or "M" or "F" or "S" or "T" or "W" or "Y" or
"V"VARIANT(14)..(14)/replace="R" or "N" or "D" or "E" or "Q" or "H"
or "I" or "L" or "K" or "M" or "F" or "S" or "T" or "W" or "Y" or
"V"VARIANT(15)..(15)/replace="Q" or
"H"VARIANT(24)..(24)/replace="R" or "N" or "D" or "E" or "Q" or "H"
or "I" or "L" or "K" or "M" or "F" or "S" or "T" or "W" or "Y" or
"V"VARIANT(27)..(27)/replace="R" or "N" or "D" or "E" or "Q" or "H"
or "I" or "L" or "K" or "M" or "F" or "S" or "T" or "W" or "Y" or
"V"VARIANT(28)..(28)/replace="Q" or
"E"VARIANT(31)..(31)/replace="R" or "N" or "D" or "E" or "Q" or "H"
or "I" or "L" or "K" or "M" or "F" or "S" or "T" or "W" or "Y" or
"V"VARIANT(34)..(34)/replace="R" or "N" or "D" or "E" or "Q" or "H"
or "I" or "L" or "K" or "M" or "F" or "S" or "T" or "W" or "Y" or
"V"VARIANT(35)..(35)/replace="Q" or
"H"MISC_FEATURE(1)..(35)/note="Variant residues given in the
sequence have no preference with respect to those in the
annotations for variant positions" 9Asp Glu Leu Ala Gln Leu Ala Arg
Glu Leu Ala Lys Leu Ala Arg Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Ala Ala Leu Ala Arg Lys Leu Ala Met 20 25 30Leu Ala Arg
351035PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 10Asp Glu Leu Ala Gln Leu Leu Glu
Glu Leu Phe Lys Leu Glu Arg Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Ile Ala Leu Trp Arg Lys Leu Ser Met 20 25 30Leu Trp Gln
351135PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 11Asp Glu Leu Ala Gln Leu Gln Glu
Glu Leu Gln Lys Leu His Gln Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Leu Ala Leu Asn Arg Lys Leu Arg Met 20 25 30Leu Gln Arg
351235PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 12Asp Glu Leu Asp Gln Leu Phe Glu
Glu Leu Arg Lys Leu Asn Arg Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Ser Ala Leu Arg Glu Lys Leu Glu Met 20 25 30Leu Leu Gln
351335PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 13Asp Glu Leu Asp Gln Leu His Glu
Glu Leu Asp Lys Leu Phe Gln Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Ser Ala Leu Ser Glu Lys Leu Lys Met 20 25 30Leu Ser Gln
351435PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 14Asp Glu Leu Glu Gln Leu Ala Arg
Glu Leu Ala Lys Leu Tyr Arg Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Glu Ala Leu Trp Glu Lys Leu Gln Met 20 25 30Leu Arg Gln
351535PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 15Asp Glu Leu Glu Gln Leu Asp Gln
Glu Leu His Lys Leu Tyr Gln Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Val Ala Leu Ser Arg Lys Leu Asp Met 20 25 30Leu Ile Arg
351635PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 16Asp Glu Leu Phe Gln Leu Ala Glu
Glu Leu Lys Lys Leu Asp His Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Ile Ala Leu Arg Gln Lys Leu Arg Met 20 25 30Leu Phe His
351735PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 17Asp Glu Leu Phe Gln Leu Thr Gln
Glu Leu Ala Lys Leu Tyr His Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Asp Ala Leu His Glu Lys Leu Glu Met 20 25 30Leu Asn Gln
351835PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 18Asp Glu Leu His Gln Leu Glu Glu
Glu Leu Glu Lys Leu Ser Gln Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Ile Ala Leu Ile Arg Lys Leu Ile Met 20 25 30Leu Met Arg
351935PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 19Asp Glu Leu His Gln Leu His Glu
Glu Leu Ile Lys Leu Ile His Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Asn Ala Leu Trp Arg Lys Leu Glu Met 20 25 30Leu His His
352035PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 20Asp Glu Leu Ile Gln Leu Ala Glu
Glu Leu Thr Lys Leu His His Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Ala Ala Leu Met Glu Lys Leu Lys Met 20 25 30Leu Arg Gln
352135PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 21Asp Glu Leu Ile Gln Leu Phe Gln
Glu Leu Phe Lys Leu Ser His Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Thr Ala Leu Lys Arg Lys Leu Phe Met 20 25 30Leu Lys His
352235PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 22Asp Glu Leu Lys Gln Leu Asn Glu
Glu Leu Glu Lys Leu Val Gln Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Gln Ala Leu Asp Arg Lys Leu Ala Met 20 25 30Leu Asn Gln
352335PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 23Asp Glu Leu Lys Gln Leu Tyr Gln
Glu Leu Val Lys Leu His His Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Ala Ala Leu Trp Arg Lys Leu Asn Met 20 25 30Leu Tyr Arg
352435PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 24Asp Glu Leu Leu Gln Leu Asp Glu
Glu Leu Ser Lys Leu Met Arg Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Asp Ala Leu Gln Glu Lys Leu Thr Met 20 25 30Leu Glu His
352535PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 25Asp Glu Leu Leu Gln Leu Ile Glu
Glu Leu Thr Lys Leu Arg His Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Met Ala Leu Ala Arg Lys Leu His Met 20 25 30Leu Thr Gln
352635PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 26Asp Glu Leu Met Gln Leu Asp Glu
Glu Leu Lys Lys Leu Thr His Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Leu Ala Leu His Glu Lys Leu Ser Met 20 25 30Leu Tyr His
352735PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 27Asp Glu Leu Met Gln Leu His Gln
Glu Leu Lys Lys Leu Gln His Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Phe Ala Leu Leu Glu Lys Leu Met Met 20 25 30Leu Trp Arg
352835PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 28Asp Glu Leu Asn Gln Leu Ala Glu
Glu Leu Lys Lys Leu Thr Gln Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Met Ala Leu Gln Glu Lys Leu Asn Met 20 25 30Leu Phe Gln
352935PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 29Asp Glu Leu Gln Gln Leu Lys Gln
Glu Leu Arg Lys Leu Lys Gln Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Thr Ala Leu Glu Arg Lys Leu Ile Met 20 25 30Leu Thr Arg
353035PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 30Asp Glu Leu Gln Gln Leu Leu Glu
Glu Leu Asp Lys Leu Met Gln Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Glu Ala Leu Gln Gln Lys Leu Glu Met 20 25 30Leu Trp Gln
353135PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 31Asp Glu Leu Arg Gln Leu Ala Glu
Glu Leu Val Lys Leu Glu Gln Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Gln Ala Leu Arg Gln Lys Leu Leu Met 20 25 30Leu Thr Gln
353235PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 32Asp Glu Leu Arg Gln Leu Asp Glu
Glu Leu Lys Lys Leu Thr His Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Arg Ala Leu Tyr Glu Lys Leu Asp Met 20 25 30Leu Ala His
353335PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 33Asp Glu Leu Ser Gln Leu Asp Glu
Glu Leu Gln Lys Leu Ser Arg Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Glu Ala Leu Met Glu Lys Leu Ser Met 20 25 30Leu Met His
353435PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 34Asp Glu Leu Ser Gln Leu Glu Glu
Glu Leu Phe Lys Leu His Arg Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Asp Ala Leu Glu Glu Lys Leu Ile Met 20 25 30Leu Val His
353535PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 35Asp Glu Leu Thr Gln Leu Ala Glu
Glu Leu Ser Lys Leu Glu Gln Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Ile Ala Leu Ile Arg Lys Leu His Met 20 25 30Leu Thr Gln
353635PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 36Asp Glu Leu Thr Gln Leu Lys Glu
Glu Leu Lys Lys Leu Leu His Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Trp Ala Leu Leu Glu Lys Leu Arg Met 20 25 30Leu Met Gln
353735PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 37Asp Glu Leu Val Gln Leu Asn Glu
Glu Leu Lys Lys Leu Phe His Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Met Ala Leu Ile Glu Lys Leu Lys Met 20 25 30Leu Arg Gln
353835PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 38Asp Glu Leu Trp Gln Leu Ala Glu
Glu Leu Arg Lys Leu Met Arg Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Met Ala Leu Trp Gln Lys Leu Val Met 20 25 30Leu Met Gln
353935PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 39Asp Glu Leu Trp Gln Leu His Glu
Glu Leu Asp Lys Leu Lys Arg Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Gln Ala Leu Val Glu Lys Leu Lys Met 20 25 30Leu Met His
354035PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 40Asp Glu Leu Tyr Gln Leu Glu Gln
Glu Leu Asn Lys Leu Phe His Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Lys Ala Leu Ile Arg Lys Leu Val Met 20 25 30Leu Asp His
354135PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 41Asp Glu Leu Tyr Gln Leu Phe Gln
Glu Leu Leu Lys Leu Leu His Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Asn Ala Leu Glu Gln Lys Leu Trp Met 20 25 30Leu Val Arg
35426PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic 6xHis tag" 42His His His His His His1
54335PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 43Asp Glu Leu Met Gln Leu Met Glu
Glu Leu Val Lys Leu Asn Gln Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Trp Ala Leu His Gln Lys Leu Leu Met 20 25 30Leu Ser His
354435PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 44Asp Glu Leu Ser Gln Leu Met Glu
Glu Leu Glu Lys Leu Asn Arg Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Trp Ala Leu His Gln Lys Leu Leu Met 20 25 30Leu Thr His
354535PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 45Asp Glu Leu Ser Gln Leu Met Gln
Glu Leu Thr Lys Leu Asn Gln Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Tyr Ala Leu His Gln Lys Leu Leu Met 20 25 30Leu Thr His
354635PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 46Asp Glu Leu His Gln Leu Ala Glu
Glu Leu Trp Lys Leu Asn Gln Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Glu Ala Leu His Gln Lys Leu Leu Met 20 25 30Leu Thr His
354735PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 47Asp Glu Leu Ala Gln Leu Ala Glu
Glu Leu Arg Lys Leu Asn Gln Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Trp Ala Leu His Gln Lys Leu Leu Met 20 25 30Leu Ser His
354835PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 48Asp Glu Leu Phe Gln Leu Ala Glu
Glu Leu Lys Lys Leu Asn Gln Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu
Ala Ala Leu His Gln Lys Leu Leu Met 20 25 30Leu Thr His
354935PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 49Asp Glu Leu Leu Gln Leu Ala Glu
Glu Leu Asp Lys Leu Asn Arg Gln1 5 10
15Gly Val Asp Ser Asp Glu Leu Phe Ala Leu His Gln Lys Leu Leu Met
20 25 30Leu Thr His 355035PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 50Asp Glu Leu Thr Gln Leu Ala Glu Glu Leu Val Lys Leu
Asn Gln Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu Phe Ala Leu His Gln
Lys Leu Leu Met 20 25 30Leu Thr His 355135PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 51Asp Glu Leu Tyr Gln Leu Ile Glu Glu Leu Glu Lys Leu
Asn Arg Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu Ala Ala Leu His Gln
Lys Leu Leu Met 20 25 30Leu Thr His 355235PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 52Asp Glu Leu Asp Gln Leu Ile Glu Glu Leu Thr Lys Leu
Asn Arg Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu Phe Ala Leu His Gln
Lys Leu Leu Met 20 25 30Leu Ser His 355335PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 53Asp Glu Leu Leu Gln Leu Ser Glu Glu Leu Tyr Lys Leu
Asn Arg Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu Trp Ala Leu His Gln
Lys Leu Leu Met 20 25 30Leu Ser His 355435PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 54Asp Glu Leu Asn Gln Leu Met Glu Glu Leu Val Lys Leu
Asn Gln Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu Trp Ala Leu His Gln
Lys Leu Leu Met 20 25 30Leu Ser His 355535PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 55Asp Glu Leu Arg Gln Leu Met Glu Glu Leu Lys Lys Leu
Asn Gln Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu Gln Ala Leu His Gln
Lys Leu Leu Met 20 25 30Leu Thr His 355635PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 56Asp Glu Leu Ala Gln Leu Ala Glu Glu Leu Arg Lys Leu
Asn Gln Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu Tyr Ala Leu His Gln
Lys Leu Leu Met 20 25 30Leu Ser His 355735PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 57Asp Glu Leu Ala Gln Leu Ile Glu Glu Leu Tyr Lys Leu
Glu Gln Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu Asp Ala Leu His Gln
Lys Leu Ile Met 20 25 30Leu Tyr Arg 355835PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 58Asp Glu Leu Asp Gln Leu Glu Glu Glu Leu Trp Lys Leu
Asn Gln Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu Glu Ala Leu His Gln
Lys Leu Leu Met 20 25 30Leu Thr His 355935PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 59Asp Glu Leu Leu Gln Leu Ser Glu Glu Leu Tyr Lys Leu
Asn Arg Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu Tyr Ala Leu His Gln
Lys Leu Leu Met 20 25 30Leu Thr His 356035PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 60Asp Glu Leu Met Gln Leu Phe Glu Glu Leu Thr Lys Leu
Asn Gln Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu Tyr Ala Leu His Gln
Lys Leu Leu Met 20 25 30Leu Thr His 356135PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 61Asp Glu Leu Met Gln Leu Met Glu Glu Leu Val Lys Leu
Asn Gln Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu Phe Ala Leu His Gln
Lys Leu Leu Met 20 25 30Leu Thr His 356235PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 62Asp Glu Leu Gln Gln Leu Lys Glu Glu Leu Thr Lys Leu
Asn Gln Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu Tyr Ala Leu His Gln
Lys Leu Leu Met 20 25 30Leu Tyr His 356335PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 63Asp Glu Leu Ser Gln Leu Met Glu Glu Leu Glu Lys Leu
Asn Arg Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu Ala Ala Leu His Gln
Lys Leu Leu Met 20 25 30Leu Thr His 356435PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 64Asp Glu Leu Ser Gln Leu Met Glu Glu Leu Glu Lys Leu
Asn Arg Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu Phe Ala Leu His Gln
Lys Leu Leu Met 20 25 30Leu Ser His 356535PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 65Asp Glu Leu Thr Gln Leu Ala Glu Glu Leu Lys Lys Leu
Asn Gln Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu Ala Ala Leu His Gln
Lys Leu Leu Met 20 25 30Leu Ser His 356635PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 66Asp Glu Leu Val Gln Leu Arg Gln Glu Leu Ala Lys Leu
Ile His Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu Glu Ala Leu Tyr Arg
Lys Leu Met Met 20 25 30Leu Glu Gln 356735PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 67Asp Glu Leu Tyr Gln Leu Ile Glu Glu Leu Glu Lys Leu
Asn Arg Gln1 5 10 15Gly Val Asp Ser Asp Glu Leu Ala Ala Leu His Gln
Lys Leu Leu Met 20 25 30Leu Ser His 35
* * * * *